Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells

  • Hendrik Schmidt1,

    Affiliated with

    • Christoph Gelhaus2,

      Affiliated with

      • Melanie Nebendahl1,

        Affiliated with

        • Marcus Lettau1,

          Affiliated with

          • Ralph Lucius3,

            Affiliated with

            • Matthias Leippe2,

              Affiliated with

              • Dietrich Kabelitz1 and

                Affiliated with

                • Ottmar Janssen1Email author

                  Affiliated with

                  Cell Communication and Signaling20119:4

                  DOI: 10.1186/1478-811X-9-4

                  Received: 1 December 2010

                  Accepted: 21 January 2011

                  Published: 21 January 2011

                  Abstract

                  Background

                  Cytotoxic cells of the immune system have evolved a lysosomal compartment to store and mobilize effector molecules. In T lymphocytes and NK cells, the death factor FasL is one of the characteristic marker proteins of these so-called secretory lysosomes, which combine properties of conventional lysosomes and exocytotic vesicles. Although these vesicles are crucial for immune effector function, their protein content in T cells has so far not been investigated in detail.

                  Results

                  In the present study, intact membranous vesicles were enriched from homogenates of polyclonally activated T cells and initially characterized by Western blotting and electron microscopic inspection. The vesicular fraction that contained the marker proteins of secretory lysosomes was subsequently analyzed by 2D electrophoresis and mass spectrometry. The proteome analysis and data evaluation revealed that 70% of the 397 annotated proteins had been associated with different lysosome-related organelles in previous proteome studies.

                  Conclusion

                  We provide the first comprehensive proteome map of T cell-derived secretory lysosomes with only minor contaminations by cytosolic, nuclear or other proteins. This information will be useful to more precisely address the activation-dependent maturation and the specific distribution of effector organelles and proteins in individual T or NK cell populations in future studies.

                  Background

                  Cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells are the main cytotoxic effector cells of the immune system. In order to effectively eliminate virus-infected and tumorigenic cells, they rapidly mobilize effector molecules including granzymes, perforin, granulysin and the death factor FasL (CD178) that are presumably stored in preformed organelles termed secretory lysosomes (SL) [1]. Secretory lysosomes combine degradative properties of conventional lysosomes with characteristics of exocytotic vesicles. At the level of morphology, conventional and secretory lysosomes are hardly distinguishable and both appear to represent endpoints of an endocytotic pathway and are formed by fusion and fission of endosomes and lysosomes [2]. Similar to conventional lysosomes, large membrane areas are covered by lysosome-associated membrane-proteins (LAMPs) including LAMP-1 (CD107a), LAMP-2 (CD107b) and LAMP-3 (CD63) [35]. However, secretory effector lysosomes are characterized by a specific set of membrane and luminal marker proteins [6, 7]. The current consensus is that SL of CTLs and NK cells carry the aforementioned effector proteins either in the lysosomal lumen (granzymes, perforin and granulysin) or as characteristic transmembrane compounds (FasL) [810].

                  Recently, we provided a protocol that allows a substantial enrichment of intact SL from in vitro expanded lymphocyte populations [11]. Employing this procedure for subcellular fractionation of a crude organelle preparation, we obtained a fraction of intact vesicles that is significantly enriched in SL marker proteins. We were thus able to report the first comprehensive analysis of the luminal proteome of secretory lysosomes from NK cells [12]. At that time, 234 different proteins were identified by mass spectrometry, 77% of which had been associated with SL or other lysosomal compartments before. Applying 2D difference gel electrophoresis, we also described a cell line-specific distribution of functionally relevant proteins in SL from human NK cell lines and primary NK cells [12].

                  Based on this study, it appears likely that different T cell populations utilize the SL organelles to store and mobilize lineage-specific cargo proteins. However, the proteome of secretory lysosomes in T cells has not been deciphered. To provide the first proteome map for T cell-derived SL, we enriched organelles from activated T lymphoblasts. Organelle extracts were subjected to SDS-PAGE and Western blotting to identify the FasL-containing SL fraction. This fraction was analyzed by electron microscopy to demonstrate the enrichment of a homogeneous population of intact vesicles. In order to define the luminal proteome of the respective SL compartment, the organelles were lysed and proteins were separated by 2D gel electrophoresis. Mass spectrometry was applied to identify individual spots. We annotated 397 proteins, 70% of which had been associated with lysosome-related organelles before. With the present report, we thus provide the first comprehensive description of the content of FasL-carrying effector vesicles isolated from activated human T lymphocytes.

                  Results and Discussion

                  In our preceding analysis of the SL compartment of NK cell lines and primary NK cells, we annotated 234 individual proteins and demonstrated a cell line-specific distribution of several functionally relevant molecules including cytotoxic effector proteins, lysosomal proteases and MHC molecules [12]. As a basis to address unsolved issues regarding the maturation, function and cell type-specific composition of the cytotoxic effector compartment in T cell populations, we now analyzed the proteome of enriched secretory lysosomes from in vitro activated human T cell blasts.

                  FasL-associated secretory lysosomes in activated lymphocytes

                  We and others have shown that in CTLs, preformed FasL accumulates in the limiting membrane of secretory lysosomes with late endosome or multi-vesicular-body structure and there co-localizes with characteristic lysosomal marker proteins including CD63 or lysosomal hydrolases and cytoskeletal adapter proteins [79, 1315]. Confocal laser-scanning microscopy (CLSM) was applied to confirm that FasL also might serve as a marker for secretory lysosomes in in vitro expanded PHA-stimulated T lymphocytes used in the present study. As depicted in Figure 1, we detected an apparent co-localization of CD63 with FasL, granzyme A and the lysosomal protease cathepsin B. It should be mentioned that a common or distinct localization of LAMP-3 (CD63) and FasL is still controversially discussed. Several reports suggest a co-localization of FasL with granule proteins, such as cathepsin D, CD63, granzyme B, perforin and LAMP-1 in a single granular entity [8, 9] whereas other studies indicate that CD63 and FasL are located in distinct subcellular compartments [16].
                  http://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_219_Fig1_HTML.jpg
                  Figure 1

                  In T cell blasts, FasL associates with lysosomal vesicles. PHA blasts (d14) were fixed and stained for FasL with NOK1 and Alexa Fluor488-conjugated donkey anti-mouse IgG or for granzyme A with GrA-11 FITC-conjugated mAbs or for cathepsin B with polyclonal goat anti-cathepsin B (N-19) antibodies and Alexa Fluor488-conjugated donkey anti-goat IgG. After extensive washing, all samples were stained for CD63 with Alexa Fluor555-conjugated mAb MEM-259. Nuclei were visualized by DAPI (bar: 10 μm).

                  Our protocol for the enrichment of secretory lysosomes yielded six separate fractions that were subjected to further analysis by Western blotting or 2D gel electrophoresis. To demonstrate an effective enrichment of the SL fraction, we first separated the proteins of individual fractions by SDS-PAGE and stained for characteristic organelle marker proteins after Western blotting. As shown in Figure 2, indicated by the high abundance of FasL, CD63 and cathepsin D, SL were enriched in fraction 2. Although LAMP-1 was also enriched in this fraction, the presence of this lysosomal membrane protein in other fractions might indicate the complex composition of the lysosomal compartment in general and that other lysosome-related vesicles might exist with distinct biophysical properties that separate at different media densities. As further indicators for the effective organelle enrichment and separation, we used cytochrome oxidase subunit IV (CoxIV) as a marker for mitochondria (see enriched organelles and fraction 5 in Figure 2) and pan-cadherin as a marker for the plasma membrane (only present in whole cell lysates). Of note, all proteins that were enriched in separate fractions were of course also present in the enriched organelle (EO) fraction placed on the gradient. However, due to the the relatively low abundance of individual proteins in the EO fraction, Western blot detection at the displayed exposure time did only reveal very faint bands. This is in agreement with our previous report [11] in which we showed a massive enrichment of FasL in fraction 2 while in the starting EO material from different T cell populations, FasL was almost not detectable at the same exposure time.
                  http://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_219_Fig2_HTML.jpg
                  Figure 2

                  Western blot analysis of subcellular fractions. Individual lanes represent whole cell lysate (WL), enriched organelles (EO) and the six fractions collected after density gradient centrifugation of IL-2 expanded T lymphocytes. The blots were probed with antibodies against respective markers for lysosomal (CD63, LAMP-1, cathepsin D) and secretory organelles (FasL). Pan-cadherin served as a marker for plasma membranes, CoxIV for mitochondria.

                  Regarding the "purity" of the obtained fraction, it should be stressed that most if not all enrichment protocols published so far do not allow a "purification" rather than an "enrichment" of a given organelle population. This is presumably based on the fact that lysosome formation and protein loading is a highly dynamic process that implies fusion and fission of several membraneous compartments and a complex protein sorting and transport machinery. For the initial characterization of enriched SL [11], we already pointed to potential "contaminations" in fraction 2, using antibodies against EEA1, a putative marker for endosomes, or Bip/Grp78, a marker for ER, respectively. Interestingly, during these analyses, golgin, a marker for the golgi apparatus/cisternae was only detected in fractions 3-6, but not in fractions 1 and 2 [11]. For the present study, we thus restricted ourselves to routinely check for the marker proteins depicted in Figure 2.

                  The enriched SL fraction consists of homogeneous intact vesicles

                  In addition to the biochemical analysis of the individual fractions, we visualized the obtained lysosomal fraction 2 by electron microscopy in comparison to the putative mitochondrial fraction 5. Figure 3 provides characteristic overview pictures of the two fractions. In both cases, the organelles within one fraction display a high degree of homogeneity with respect to their morphology (Figure 3A,C). At higher magnification, the characteristics of the organelles in fraction 2 become apparent. These membranous vesicles are round-shaped with a maximum size of about 700 nm and display a characteristic electron density. In contrast, organelles of fraction 5 are characterized by irregular internal membranous structures (Figure 3B,D) as expected for mitochondria.
                  http://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_219_Fig3_HTML.jpg
                  Figure 3

                  Electron micrographs of fractions 2 and 5. Enriched organelles from PHA blasts corresponding to fractions 2 (A, B) and 5 (C, D) were examined under an electron microscope. Overview pictures are given in A and C, magnified areas are shown in B and D. (scale as indicated)

                  The luminal proteome of enriched SL as analyzed by 2D-PAGE and mass spectrometry

                  In order to obtain a comprehensive list of putative luminal proteins of secretory lysosomes, enriched fraction 2 vesicles of PHA-stimulated T lymphoblasts were subjected to 2D-PAGE. More than 1600 spots from 6 replicate gels were subsequently subjected to proteolytic cleavage and lead to the mass spectrometric identification of 1335 spots. Due to repetitive identifications at respective spot locations in different gels, the actual number of identified individual spots decreased to 742. The resulting proteome map is shown as an overview in Figure 4. Additional information on identified proteins and images of individual quadrants to match proteins to respective spots are given as additional files 1, 2 and 3 (Table S1, FigureS1, Dataset S1). Multiple (up to six) identifications in separate gels from individual secretory lysosomes preparations from T cells of different donors also underscore the reproducibility of the isolation protocol [11]. Overall, the identified spots represent a total of 397 separate protein entries in the NCBI database that are listed according to their protein names, the predicted subcellular distribution and function in Table 1.
                  http://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_219_Fig4_HTML.jpg
                  Figure 4

                  2D proteome map of enriched secretory lysosomes from activated T cell blasts (overview). A total of 250 μg of fraction 2 protein were separated on pH 3-11NL IPG strips in the first and on 12.5% Tris-glycin gels in the second dimension. Proteins stained by Flamingo Pink were detected using fluorescence imaging. From a total of six gels, 742 spots were identified and annotated as 397 individual proteins. Enlarged sections of all four quadrants are available as additional file to allow the positioning of individual annotations given in table 1.

                  Table 1

                  Proteins identified in enriched secretory lysosomes from activated T cells.

                  Protein name

                  Spot #

                  Predicted/annotated subcellular localisation

                  Predicted function

                  100 kDa coactivator

                  122

                  ER,ME,PL

                  biosynthesis

                  14-3-3 protein beta

                  714

                  CY,ME

                  adapter

                  14-3-3 protein epsilon

                  685

                  CY,ME

                  adapter

                  14-3-3 protein zeta/delta

                  708

                  CY,ME

                  adapter

                  2',3'-cyclic-nucleotide 3'-phosphodiesterase

                  542

                  ME,NG

                  hydrolase

                  26S protease regulatory subunit 6A

                  1037

                  CY,NU

                  protein degradation

                  26S proteasome non-ATPase regulatory subunit 2

                  165

                  ME

                  proteasome

                  3-phosphoglycerate dehydrogenase

                  408

                  ME

                  biosythesis

                  acetyl-CoA acetyltransferase, cytosolic

                  594

                  CY

                  biosynthesis

                  ACTB protein

                  525

                  ER,ME,EX,PL,SY

                  cell motility

                  actin related protein 2 isoform b

                  564

                  EN,ER,ME

                  trafficking

                  actin related protein 2/3 complex subunit 1B

                  590

                  ME,PL

                  trafficking

                  actin related protein 2/3 complex subunit 2

                  674

                  EN,ME,PL,ER

                  trafficking

                  actin related protein 2/3 complex subunit 3

                  799

                  EN,ME,PL,ER

                  trafficking

                  actin related protein 2/3 complex subunit 4

                  798

                  ER,EX,ME,SY

                  trafficking

                  actin related protein 2/3 complex subunit 5-like

                  804

                  ME

                  trafficking

                  actin, alpha, cardiac muscle

                  570

                  ME

                  cell motility

                  actin, gamma 1 propeptide

                  701

                  ME,EX,PL,SY

                  cell motility

                  actinin, alpha 4

                  144

                  ME,NU,CY

                  cell motility

                  acylamino acid-releasing enzyme

                  203

                  PL, CY

                  hydrolase

                  acyl-CoA synthetase long-chain family member 4 isoform 2

                  199

                  MT,PE,ME

                  metabolism

                  adenine phosphoribosyltransferase isoform b

                  784

                  ME,EX,PL

                  biosynthesis

                  adenosine deaminase

                  550

                  CY,LY

                  hydrolase

                  adenylosuccinate lyase

                  439

                  CY,PL

                  biosynthesis

                  adenylosuccinate synthetase

                  1023

                  CY

                  biosynthesis

                  adenylyl cyclase-associated protein variant

                  395

                  PL,ME

                  trafficking

                  aflatoxin aldehyde reductase AFAR

                  1056

                  PL,GO

                  redox protein

                  aging-associated gene 12

                  418

                  unknown

                  unclassified

                  alanyl-tRNA synthetase

                  111

                  ME,PL

                  biosynthesis

                  alcohol dehydrogenase class-3

                  587

                  PL,CY

                  redox protein

                  aldo-keto reductase family 1, member A1

                  1078

                  PL,SY

                  metabolism

                  aldolase A

                  560

                  EN,ME

                  metabolism

                  aldose 1-epimerase (BLOCK25)

                  596

                  CY

                  metabolism

                  aldose reductase

                  981

                  ME,EX,MT

                  metabolism

                  alkyldihydroxyacetonephosphate synthase, peroxisomal

                  273

                  PE

                  biosynthesis

                  alpha-tubulin

                  362

                  PL

                  cell motility

                  annexin A1

                  615

                  ME,MT

                  trafficking

                  annexin A11

                  415

                  MT,EX,ME

                  trafficking

                  annexin A2

                  631

                  MT,EX,ME,secreted

                  exocytosis

                  annexin A4

                  668

                  MT,EX,ME,SY

                  signal trans.

                  annexin A5

                  660

                  MT,EX,ME,ER

                  trafficking

                  annexin A6

                  263

                  MT,EX,ME,ER

                  trafficking

                  annexin A7 isoform 2

                  504

                  EX,ME,PL

                  exocytosis

                  ARP3 actin-related protein 3 homolog

                  465

                  EN,ER,ME

                  trafficking

                  ARTS-1

                  136

                  ER,ME

                  immunity

                  aryl hydrocarbon receptor interacting protein

                  955

                  unknown

                  unclassified

                  asparaginyl-tRNA synthetase

                  306

                  ME

                  biosynthesis

                  aspartate aminotransferase

                  575

                  CY

                  biosynthesis

                  ATP citrate lyase

                  96

                  ME,EX,PL

                  biosynthesis

                  ATP synthase, alpha subunit precursor

                  426

                  ER,LY,NG,SY,MT

                  channel

                  ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B2

                  413

                  LY,ME,NG,SY

                  channel

                  axin interactor, dorsalization associated protein

                  632

                  unknown

                  signal trans.

                  beta adrenergic receptor kinase 1

                  220

                  CY

                  GTPase

                  bifunctional purine biosynthesis protein PURH

                  326

                  ME,PL

                  mutifunctional

                  bleomycin hydrolase

                  463

                  CY,PL

                  hydrolase

                  BolA-like protein 2

                  867

                  ME

                  unclassified

                  calcium binding protein 39

                  622

                  EX

                  unclassified

                  CALM3 protein

                  816

                  unknown

                  unclassified

                  calreticulin precursor variant

                  287

                  ER,ME,NG,EX,PL,MT

                  chaperone

                  carboxyl terminal LIM domain protein

                  612

                  ER,ME,PL,EN

                  cell motility

                  catalase

                  361

                  PE,ER,LY,EN,ME

                  metabolism

                  cathepsin B

                  691

                  LY,ME,NG

                  hydrolase

                  cathepsin D preproprotein

                  696

                  LY,ME,NG,EX,MT

                  hydrolase

                  cathepsin H

                  738

                  LY

                  hydrolase

                  cathepsin S

                  749

                  LY

                  immunity

                  Cbr1 In Complex With Hydroxy-Pp

                  666

                  ME

                  redox protein

                  Cdc42ACK GTPASE

                  790

                  ME

                  cell motility

                  centaurin beta1

                  214

                  unknown

                  GTPase

                  chaperonin (HSP60)

                  338

                  ME,NG,EX,SY,MT

                  chaperone

                  chaperonin containing TCP1, subunit 2β

                  424

                  ER,EN,ME,PL,MT,CY

                  chaperone

                  chaperonin containing TCP1, subunit 7η

                  376

                  EN,ME,PL

                  chaperone

                  chaperonin containing TCP1, subunti 5ε

                  339

                  EX,PL

                  chaperone

                  chaperonin containing TCP1, subunti 8τ

                  357

                  EN,ME,EX

                  chaperone

                  chaperonin containing TCP1, subunti 8τ

                  358

                  EN,ME,EX

                  chaperone

                  chromatin modifying protein 4B

                  639

                  ME,EX

                  trafficking

                  chromosome 20 open reading frame 3 (BSCv)

                  489

                  ME

                  unclassified

                  chromosome 9 open reading frame 19

                  831

                  EX,GO

                  unclassified

                  N2 protein

                  651

                  unknown

                  unclassified

                  coactosin-like protein

                  841

                  PL,SY

                  unclassified

                  cofilin 1

                  810

                  ER,ME,EX,MT

                  cell motility

                  copine I

                  310

                  ME

                  trafficking

                  copine III

                  340

                  ME,EX,PL

                  trafficking

                  coronin 7

                  113

                  CY,GO

                  trafficking

                  coronin, actin binding protein, 1A

                  371

                  LY,PL

                  cell motility

                  coronin, actin binding protein, 1C

                  353

                  ME

                  multifunctional

                  c-src tyrosine kinase

                  475

                  CY,PL

                  signal trans.

                  cyclophilin A

                  826

                  ME,EX,MT

                  chaperone

                  cyclophilin B

                  800

                  ER,ME

                  chaperone

                  cystatin B

                  857

                  ME

                  protein inhibitor

                  cysteine and glycine-rich protein 1

                  769

                  NU

                  unclassified

                  cytoskeleton associated protein

                  952

                  CY

                  cell motility

                  cytosolic malate dehydrogenase

                  642

                  ME,EX,PL,SY,MT

                  metabolism

                  DCHT2 Serine/threonine-protein kinase OSR1

                  332

                  ME

                  signal trans.

                  destrin isoform a

                  814

                  ME,EX,MT

                  cell motility

                  differentially expressed in FDCP 6 homolog (mouse), isoform CRA_b

                  228

                  unknown

                  unclassified

                  dihydropyrimidinase-like 2

                  319

                  SY

                  signal trans.

                  dimethylarginine dimethylaminohydrolase 2

                  683

                  unknown

                  hydrolase

                  dipeptidyl peptidase 4

                  81

                  ER,LY,EN,ME,EX

                  hydrolase

                  DJ-1 protein

                  764

                  ME,PL,SY,MT

                  redox protein

                  DnaJ (Hsp40) homolog, subfamily A, member 1, isoform CRA_d

                  499

                  ER,ME

                  chaperone

                  DnaJ (Hsp40) homolog, subfamily B, member 11 precursor

                  551

                  ER

                  chaperone

                  docking protein 2

                  455

                  unknown

                  unclassified

                  dynamin 2 isoform 1

                  148

                  EN

                  trafficking

                  echinoderm microtubule associated protein like 2 variant

                  929

                  CY

                  cell motility

                  EF-hand domain family, member D2

                  1012

                  unknown

                  unclassified

                  EH-domain containing 1

                  342

                  ER,LY,EN,EX,PL

                  trafficking

                  Ena-VASP-like protein

                  447

                  CY

                  cell motility

                  ENC-1AS aka Beta-hexosaminidase subunit beta

                  431

                  LY

                  multifunctional

                  endoplasmic reticulum protein 29 isoform 1 precursor

                  721

                  ER,ME,PL

                  chaperone

                  enolase 1 variant

                  496

                  ME,EX,SY,MT

                  metabolism

                  ERAP2 protein

                  99

                  ER

                  immunity

                  ERBB2IP protein

                  197

                  NU,CY

                  multifunctional

                  ERO1L

                  311

                  ME,ER

                  redox protein

                  esterase D/formylglutathione hydrolase

                  656

                  ME

                  hydrolase

                  eukaryotic translation elongation factor 1 alpha 1

                  462

                  ER,LY,EN,ME,EX,PL

                  biosynthesis

                  eukaryotic translation elongation factor 1 gamma, isoform CRA_d

                  947

                  ME

                  biosynthesis

                  eukaryotic translation elongation factor 2

                  158

                  ER,EN,ME,EX

                  biosynthesis

                  eukaryotic translation initiation factor 4A

                  505

                  ME

                  biosynthesis

                  eukaryotic translation initiation factor 5A

                  817

                  ME

                  biosynthesis

                  extended-synaptotagmin-1 KIAA0747 protein

                  155

                  ME

                  unclassified

                  ezrin

                  208

                  CY

                  cell motility

                  F-actin capping protein alpha-1 subunit

                  611

                  ER,EN,ME

                  actin binding

                  F-actin capping protein alpha-1 subunit variant

                  623

                  ER,EN,ME

                  actin binding

                  F-actin capping protein alpha-2 subunit

                  616

                  ER,EN,ME,PL

                  cell motility

                  F-actin capping protein beta subunit

                  663

                  ER,EN,ME

                  actin binding

                  farnesyl pyrophosphate synthetase

                  579

                  CY

                  biosynthesis

                  FK506 binding protein 1A

                  856

                  ME,SY,MT

                  signal trans.

                  flotillin 1

                  486

                  LY,ME,EX

                  membrane

                  formin-binding protein 1

                  1059

                  SL, LY, CY

                  adapter

                  fructose-bisphosphate aldolase C

                  565

                  ME,SY,MT

                  metabolism

                  fumarate hydratase, mitochondrial

                  507

                  EN,SY,MT

                  cell cycle

                  FYN-binding protein

                  71

                  CY,NU

                  adapter

                  G protein beta subunit

                  638

                  ME,MT

                  signal trans.

                  galectin-1

                  851

                  ME,PL

                  immunity

                  galectin-3

                  718

                  ME,NU

                  immunity

                  gamma-enolase

                  476

                  ME,PL,SY

                  glycolysis

                  gamma-glutamyl hydrolase

                  629

                  LY,ME,NG,PL

                  hydrolase

                  GDP-mannose pyrophosphorylase A

                  541

                  unknown

                  biosythesis

                  gelsolin-like capping protein isoform 9

                  572

                  ME,CY,NU

                  cell motility

                  GIPC1 protein

                  598

                  SY,CY

                  protein binding

                  glia maturation factor gamma

                  818

                  unknown

                  unclassified

                  glucosamine-6-phosphate deaminase 1

                  677

                  CY

                  hydrolase

                  glucose-6-phosphate dehydrogenase isoform b

                  409

                  ME

                  metabolism

                  glucosidase II subunit beta

                  126

                  ER,ME,PL

                  hydrolase

                  glucosidase, alpha; neutral AB, isoform CRA_a

                  936

                  ER,ME,PL

                  hydrolase

                  glutamate carboxypeptidase

                  430

                  unknown

                  hydrolase

                  glutamate Dehydrogenase-Apo Form

                  437

                  ER,ME,PL,MT

                  unclassified

                  glutaredoxin 3

                  589

                  CY

                  redox protein

                  glutathione S-transferase P1

                  766

                  ER,ME,EX,PL

                  metabolism

                  glutathione synthetase

                  461

                  PL

                  redox protein

                  glutathione-S-transferase kappa 1

                  765

                  PL,ME,MT,PE

                  unclassified

                  glutathione-S-transferase omega 1

                  698

                  LY,ME,NG,EX,PL,SY,MT

                  metabolism

                  glyceraldehyde-3-phosphate dehydrogenase

                  610

                  LY,ME,NG,EX,PL,SY,MT

                  metabolism

                  glycyl-tRNA synthetase

                  244

                  ME

                  biosynthesis

                  glyoxalase domain containing 4

                  653

                  MT

                  unclassified

                  GNAS complex locus isoform f

                  531

                  EX

                  multifunctional

                  GNB1 protein

                  634

                  EN,ME,EX,PL,SY

                  signal trans.

                  granzyme A

                  724

                  SL

                  immunity

                  GRAP2 protein

                  957

                  unknown

                  unclassified

                  GRB2 protein

                  756

                  SY

                  adapter

                  GTP-binding nuclear protein Ran

                  755

                  ME,EX

                  trafficking

                  guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 2, isoform CRA_c

                  582

                  EX

                  GTPase

                  guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1, isoform CRA_d

                  664

                  ER

                  signal trans.

                  guanine nucleotide-binding protein G(k) subunit alpha

                  585

                  ME,EX

                  trafficking

                  guanine nucleotide-binding protein subunit alpha-13

                  989

                  ME

                  signal trans.

                  haloacid dehalogenase-like hydrolase domain containing 2

                  690

                  unknown

                  hydrolase

                  heat shock 70kDa protein 1A

                  278

                  ER,EN,ME,EX,MT

                  chaperone

                  heat shock 70kDa protein 5

                  226

                  ER,ME,EX,PL,MT

                  chaperone

                  heat shock 70kDa protein 8 isoform 1

                  259

                  LY,ME,NG,EX,PL,SY,MT

                  chaperone

                  heat shock 70kDa protein 8 isoform 1

                  260

                  LY,ME,NG,EX,PL,SY,MT

                  chaperone

                  heat shock protein 70

                  112

                  EX

                  chaperone

                  heat shock protein HSP 90-alpha

                  969

                  ME,NG,MT

                  chaperone

                  heat shock protein HSP 90-beta

                  177

                  ME,EX,MT

                  chaperone

                  hematopoietic cell-specific Lyn substrate 1

                  181

                  CY,MT

                  signal trans.

                  HEXA protein

                  422

                  LY

                  multifunctional

                  hexose-6-phosphate dehydrogenase

                  194

                  ER

                  metabolism

                  HIP-55

                  377

                  CY

                  signal trans.

                  histidine triad nucleotide binding protein 1

                  852

                  ME,PL,SY

                  hydrolase

                  histocompatibility (minor) HA-1

                  1072

                  unknown

                  GTPase

                  hypothetical protein

                  216

                  unknown

                  unclassified

                  hypothetical protein LOC79624

                  472

                  unknown

                  unclassified

                  hypoxia up-regulated protein 1

                  47

                  ER,PL,ME

                  chaperone

                  importin subunit beta-1

                  164

                  ME

                  trafficking

                  integrin beta-2

                  80

                  PL

                  membrane

                  interleukin-16

                  210

                  secreted

                  immunity

                  isocitrate dehydrogenase 1 (NADP+), soluble, isoform CRA_b

                  540

                  ME,EX,PL

                  redox protein

                  isocitrate dehydrogenase 2 (NADP+), mitochondrial, isoform CRA_b

                  510

                  PL,MT

                  redox protein

                  kinase/transmembrane domain fusion protein

                  1061

                  unknown

                  unclassified

                  laminin-binding protein

                  543

                  ME,ER

                  cell adhesion

                  leucine aminopeptidase 3

                  432

                  CY

                  protein degradation

                  leucine rich repeat containing 57

                  747

                  unknown

                  unclassified

                  leucine-rich repeat and calponin homology domain-containing protein 5

                  908

                  MT

                  protein binding

                  leucocyte antigen CD97

                  872

                  ME,secreted

                  cell adhesion

                  leukocyte-derived arginine aminopeptidase long form variant

                  102

                  unknown

                  hydrolase

                  leukotriene A4 hydrolase

                  309

                  CY

                  hydrolase

                  LIM and SH3 domain protein 1

                  606

                  ER,EN,ME,PL

                  adapter

                  LIM domain-containing protein 2

                  834

                  unknown

                  unclassified

                  lin 7 homolog c

                  1070

                  SY

                  exocytosis

                  L-lactate dehydrogenase

                  645

                  ME,EX,SY

                  metabolism

                  L-lactate dehydrogenase B chain

                  626

                  ME,EX,PL,SY,MT

                  redox protein

                  L-plastin

                  266

                  CY

                  actin binding

                  L-plastin variant

                  267

                  unknown

                  cell motility

                  LPXN protein

                  474

                  unknown

                  unclassified

                  lymphocyte cytosolic protein 2

                  229

                  CY

                  immunity

                  lymphocyte-specific protein 1

                  959

                  PL

                  immunity

                  lysosomal acid alpha-mannosidase

                  265

                  LY,ME

                  hydrolase

                  M2-type pyruvate kinase

                  356

                  ME,EX,SY

                  metabolism

                  Macrophage Migration Inhibitory Factor (Mif) With Hydroxphenylpyruvate

                  862

                  ME,EX,PL,SY

                  immunity

                  MAGUK p55 subfamily member 7

                  292

                  PL

                  protein binding

                  methylenetetrahydrofolate dehydrogenase 1

                  139

                  EN,ME,PL,MT

                  multifunctional

                  methylthioadenosine phosphorylase

                  697

                  CY

                  metabolism

                  MHC class I antigen

                  533

                  ME

                  immunity

                  MHC class I antigen

                  865

                  ME

                  immunity

                  MHC class II antigen

                  953

                  ME

                  immunity

                  MHC class II antigen DR alpha chain

                  1050

                  LY

                  immunity

                  MHC class II antigen DR52

                  1083

                  ME

                  immunity

                  microtubule-associated protein, RP/EB family, member 1

                  665

                  ME,PL

                  cell motility

                  mitochondrial ATP synthase, H+ transporting F1 complex beta subunit

                  443

                  MT

                  trafficking

                  mitochondrial trifunctional protein, alpha subunit precursor

                  253

                  PL, MT

                  metabolism

                  mitogen-activated protein kinase 1

                  569

                  ME,PL

                  signal trans.

                  mitogen-activated protein kinase kinase 1 interacting protein 1

                  943

                  LY

                  adapter

                  mitogen-activated protein kinase kinase 2

                  509

                  unknown

                  signal trans.

                  moesin, isoform CRA_b

                  246

                  EN,ME,EX,PL,MT

                  cell motility

                  mps one binder kinase activator-like 1B

                  758

                  unknown

                  unclassified

                  myosin IG

                  75

                  unknown

                  trafficking

                  myosin light polypeptide 6

                  830

                  ME

                  cell motility

                  NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa (NADH-coenzyme Q reductase)

                  896

                  ER,ME,MT

                  trafficking

                  NCK adaptor protein 1

                  506

                  CY,ER

                  adapter

                  NECAP endocytosis associated 2

                  1010

                  EN

                  trafficking

                  NESH protein

                  434

                  unknown

                  unclassified

                  N-ethylmaleimide-sensitive factor attachment protein, alpha

                  652

                  ME,NG,PL

                  trafficking

                  neuroblastoma RAS viral (v-ras) oncogene homolog

                  779

                  GO,CY

                  trafficking

                  neuropolypeptide h3

                  781

                  ME,EX,SY

                  protein inhibitor

                  neutrophil adherence receptor alpha-M subunit

                  36

                  membrane

                  cell adhesion

                  niban protein isoform 2

                  38

                  CY

                  signal trans.

                  NME1-NME2 protein

                  823

                  CY,NU

                  multifunctional

                  nuclear chloride channel

                  684

                  ME,EX,PL,MT

                  channel

                  nucleobindin 1 variant

                  335

                  unknown

                  unclassified

                  nucleoside phosphorylase

                  670

                  CY,PL

                  cell cycle

                  nucleosome assembly protein 1-like 1, isoform CRA_d

                  315

                  ME,PL,NU

                  cell cycle

                  Obg-like ATPase 1

                  511

                  EN,ME,PL

                  hydrolase

                  otubain 1

                  637

                  ME

                  hydrolase

                  PA2G4 protein

                  490

                  unknown

                  unclassified

                  PDCD6IP protein

                  171

                  unknown

                  unclassified

                  perforin-1

                  280

                  SL

                  immunity

                  peroxiredoxin 1

                  774

                  ER,LY,EN,ME,NG,PL,MT

                  redox protein

                  peroxiredoxin 2

                  778

                  ER,EN,ME,SY,MT

                  redox protein

                  peroxiredoxin 3

                  768

                  ME,PL,MT

                  redox protein

                  peroxiredoxin 4

                  737

                  ER,EN,ME

                  redox protein

                  peroxiredoxin 6

                  945

                  LY,ME,EX,PL,SY

                  redox protein

                  PGAM1

                  730

                  ME,EX,SY

                  metabolism

                  PHB

                  948

                  unknown

                  unclassified

                  phosphatase 2a

                  316

                  MT

                  multifunctional

                  phosphatidylinositol-5-phosphate 4-kinase, type II, alpha

                  457

                  NG,PL

                  metabolism

                  phosphofructokinase, liver

                  939

                  unknown

                  glycolysis

                  phosphofructokinase, platelet

                  196

                  ME,PL

                  glycolysis

                  phosphoglucose isomerase

                  390

                  ME,EX,PL,MT

                  multifunctional

                  phosphoglycerate kinase 1

                  537

                  ME,EX,SY,MT

                  metabolism

                  phospholipase C, delta 1 variant

                  178

                  unknown

                  signal trans.

                  phosphoribosyl pyrophosphate synthetase 1 variant

                  1080

                  unknown

                  biosynthesis

                  phosphoribosylaminoimidazole carboxylase, - succinocarboxamide synthetase, isoform CRA_b

                  523

                  EN,SY

                  multifunctional

                  phosphoribosylformylglycinamidine synthase

                  64

                  CY

                  biosynthesis

                  phosphoserine aminotransferase 1

                  988

                  ME

                  biosynthesis

                  phostensin

                  91

                  CY

                  unclassified

                  poly(A) binding protein, cytoplasmic 1, isoform CRA_c

                  256

                  ER,EN,ME,PL

                  metabolism

                  poly(rC) binding protein 1

                  1082

                  ME,CY,NU

                  unclassified

                  potassium voltage-gated channel, shaker-related subfamily, beta member 2 isoform 2

                  635

                  CY

                  channel

                  PPP5C protein

                  364

                  CY,NU

                  hydrolase

                  profilin-1

                  848

                  ME,EX,PL,MT

                  actin binding

                  programmed cell death protein 10

                  741

                  unknown

                  apoptosis

                  proline synthetase co-transcribed homolog

                  699

                  CY

                  unclassified

                  prolyl 4-hydroxylase, alpha subunit

                  337

                  ER,ME

                  redox protein

                  prolyl 4-hydroxylase, beta subunit precursor

                  348

                  ER,ME,EX,PL,MT

                  redox protein

                  prolyl endopeptidase

                  234

                  CY

                  protein degradation

                  proteasome (prosome, macropain) subunit, alpha type, 7(PSMA7)

                  729

                  CY,Proteasom

                  hydrolase

                  proteasome 26S non-ATPase subunit 13 isoform 1

                  577

                  ME

                  proteasome

                  proteasome 26S subunit, ATPase, 2

                  498

                  CY,NU

                  unclassified

                  proteasome 26S subunit, ATPase, 5

                  514

                  CY,NU

                  unclassified

                  proteasome activator complex subunit 1 isoform 1

                  703

                  PL,MT

                  immunity

                  proteasome activator complex subunit 2

                  689

                  ME

                  immunity

                  proteasome alpha 2 subunit variant

                  754

                  CY

                  hydrolase

                  proteasome subunit, alpha type, 1

                  687

                  ME

                  hydrolase

                  proteasome subunit, alpha type, 5

                  1009

                  ME

                  hydrolase

                  proteasome subunit, alpha type, 6

                  734

                  CY,NU

                  hydrolase

                  proteasome subunit, beta type, 1

                  750

                  ME,CY

                  hydrolase

                  proteasome subunit, beta type, 2

                  780

                  CY,NU

                  hydrolase

                  proteasome subunit, beta type, 4

                  944

                  CY,NU

                  hydrolase

                  proteasome subunit, beta type, 8

                  773

                  PL,CY,NU

                  immunity

                  protein ARMET

                  805

                  ME, secreted

                  unclassified

                  protein diaphanous homolog 1

                  45

                  ME

                  cell motility

                  protein disulfide isomerase-associated 4

                  1060

                  ER,ME,PL

                  chaperone

                  protein disulfide isomerase-related protein 5

                  458

                  ER,ME

                  chaperone

                  protein disulfide-isomerase A3

                  379

                  ER,LY,ME,NG,EX,PL

                  chaperone

                  protein phosphatase 1, catalytic subunit, alpha isoform 1

                  603

                  EX

                  hydrolase

                  protein phosphatase 1, catalytic subunit, beta isoform

                  617

                  ME,PL

                  hydrolase

                  protein tyrosine phosphatase 1b

                  536

                  ME,ER

                  hydrolase

                  protein tyrosine phosphatase, non-receptor type 6 isoform 1 variant

                  317

                  unknown

                  hydrolase

                  protein-tyrosine kinase fyn isoform c

                  373

                  EN,CY

                  signal trans.

                  PYD and CARD domain containing

                  771

                  CY

                  apoptosis

                  pyrophosphatase 1

                  654

                  ME,MT

                  hydrolase

                  pyruvate kinase 3 isoform 2

                  346

                  ME,EX,SY

                  metabolism

                  R33729_1 (Interleukin-25)

                  837

                  ME,secreted

                  signal trans.

                  Rab GDP dissociation inhibitor beta

                  469

                  ME,EX,PL,MT

                  GTPase

                  raftlin cell migration-inducing gene 2

                  193

                  PL

                  unclassified

                  Rap1a

                  785

                  EN,ME,MT

                  GTPase

                  Rap1-GTP-interacting adapter molecule

                  141

                  CY

                  signal trans.

                  Ras GTPase-activating-like protein IQGAP2

                  1069a

                  EN

                  signal trans.

                  related RAS viral (r-ras) oncogene homolog 2 isoform a

                  1049

                  LY,ME,EX

                  GTPase

                  Rho GDP dissociation inhibitor (GDI) alpha

                  716

                  ME,PL,MT

                  GTPase

                  Rho GDP dissociation inhibitor (GDI) beta

                  728

                  CY

                  GTPase

                  Rho GTPase activating protein 1

                  441

                  PL

                  GTPase

                  Rho GTPase-activating protein 9

                  998

                  unknown

                  GTPase

                  ribosomal protein L11

                  797

                  ribosome

                  biosynthesis

                  ribosomal protein L12

                  809

                  EN,ribosom

                  biosynthesis

                  S-adenosylhomocysteine hydrolase

                  513

                  ME

                  hydrolase

                  Sec23 homolog A

                  221

                  ER,ME,PL

                  trafficking

                  Sec23B protein

                  1001

                  EN

                  trafficking

                  septin 2

                  554

                  ME,EX,SY

                  unclassified

                  septin 7

                  484

                  ME,PL,SY

                  unclassified

                  septin-9 delta

                  558

                  ME

                  unclassified

                  septin-9 gamma

                  973

                  ME

                  unclassified

                  serine/threonine phosphatase 1 gamma

                  985

                  MT,SY

                  hydrolase

                  serine/threonine-protein kinase PAK 2

                  352

                  PL

                  signal trans.

                  serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform

                  621

                  MT

                  signal trans.

                  serine/threonine-protein phosphatase 2A regulatory subunit B

                  592

                  NU

                  signal trans.

                  serpin peptidase inhibitor,clade B,member 1

                  548

                  CY

                  protein inhibitor

                  seryl-tRNA synthetase

                  365

                  ME,PL

                  tRNA processing

                  SH2 domain protein 1A

                  840

                  CY

                  signal trans.

                  SH3-containing protein, Endophilin-B1

                  1081

                  CY,GO,MT

                  apoptosis

                  SHUJUN-1

                  795

                  CY

                  cell motility

                  signal transducer and activator of transcription 1, 91kDa, isoform CRA_d

                  188

                  CY,NU

                  signal trans.

                  similar to metallo-beta-lactamase superfamily protein

                  686

                  unknown

                  hydrolase

                  small GTP binding protein Rac2, isoform CRA_c

                  1006

                  unknown

                  signal trans.

                  soc-2 suppressor of clear homolog

                  318

                  CY

                  unclassified

                  solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regulator 1

                  460

                  ME,EX,PL

                  scaffolding

                  sorting nexin 17

                  421

                  EN,ME,PL

                  trafficking

                  sorting nexin 6

                  466

                  CY

                  trafficking

                  src kinase associated phosphoprotein 1 isoform 1

                  417

                  CY,NU

                  signal trans.

                  stathmin 1/oncoprotein 18

                  820

                  SY

                  cell motility

                  stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein)

                  323a

                  ME,PL,SY

                  chaperone

                  stromal cell-derived factor 2-like 1 precursor

                  767

                  ER

                  unclassified

                  superoxide dismutase 1, soluble

                  806

                  ME,EX,MT

                  redox protein

                  syntaxin binding protein 1

                  305

                  ME,NG,EX,PL,SY

                  trafficking

                  syntaxin binding protein 2

                  302

                  EX,PL

                  trafficking

                  syntaxin binding protein 3 variant

                  294

                  ME,PL

                  trafficking

                  talin-1

                  920

                  EN,ME,PL

                  cell motility

                  tapasin isoform 3 precursor

                  495

                  ER,ME

                  immunity

                  TC4 protein

                  736

                  NU

                  GTPase

                  T-complex polypeptide 1

                  1030

                  ER,EN,ME,EX

                  chaperone

                  T-complex protein 1 subunit gamma

                  307

                  CY

                  chaperone

                  testin isoform 1

                  456

                  unknown

                  unclassified

                  thioredoxin domain-containing protein 4 precursor

                  502

                  ER,ME,PL

                  scaffolding

                  transfer RNA-Trp synthetase

                  411

                  ME,PL

                  biosynthesis

                  transgelin-2

                  787

                  ME,MT

                  unclassified

                  transketolase

                  276

                  ME,EN

                  unclassified

                  translocon-associated protein subunit delta

                  811

                  ME,ER

                  trafficking

                  triosephosphate isomerase 1

                  742

                  ME,EX,SY,MT

                  unclassified

                  tripeptidyl-peptidase 1

                  992

                  LY,ME,NG,PL,MT

                  protein degradation

                  tropomodulin 3

                  561

                  ER,ME

                  cell motility

                  tropomyosin 3 isoform 2

                  676

                  unknown

                  unclassified

                  tropomyosin 4

                  672

                  ME

                  unclassified

                  Tu translation elongation factor, mitochondrial

                  517

                  LY,ME,PL,MT

                  biosynthesis

                  tubulin alpha 6 variant

                  363

                  ME,PL

                  cell motility

                  tubulin tyrosine ligase-like family, member 12

                  897

                  ME

                  trafficking

                  tubulin, beta

                  407

                  ME,PL,SY

                  cell motility

                  tubulin, beta polypeptide

                  433

                  ME,PL,SY

                  cell motility

                  tumor rejection antigen (gp96) 1

                  118

                  ER,ME,PL,GO

                  chaperone

                  tumor susceptibility gene 101

                  470

                  EX

                  trafficking

                  twinfilin-like protein

                  578

                  CY

                  cell motility

                  tyrosine kinase LCK

                  399

                  CY

                  signal trans.

                  tyrosine-protein phosphatase non-receptor type 6

                  325

                  CY,NU

                  signal trans.

                  tyrosyl-tRNA synthetase

                  366

                  ME,PL

                  signal trans.

                  ubiquitin associated and SH3 domain containing protein A

                  913

                  CY,NU

                  protein degradation

                  ubiquitin specific peptidase 5 isoform 2

                  154

                  LY,ME,NG

                  protein degradation

                  ubiquitin specific protease 14 isoform a

                  344

                  PL

                  protein degradation

                  ubiquitin-conjugating enzyme E2 L3

                  882

                  MT

                  protein degradation

                  ubiquitin-conjugating enzyme E2 N

                  839

                  ME,EX,MT

                  differentiation

                  ubiquitin-like modifier-activating enzyme 1

                  120

                  MT,ME

                  protein degradation

                  UDP-glucose ceramide glucosyltransferase-like 1 isoform 1

                  39

                  ER,ME

                  chaperone

                  UDP-glucose pyrophosphorylase 2 isoform b

                  442

                  EN,ME

                  metabolism

                  UNC-112 related protein 2 long form

                  971

                  PL

                  cell adhesion

                  unnamed protein product

                  706

                  unknown

                  unclassified

                  UPF0550 protein C7orf28

                  450

                  ME

                  unclassified

                  vacuolar H+-ATPase 56,000 subunit

                  414

                  LY,ME,NG,SY

                  channel

                  vacuolar protein sorting 45A

                  322

                  LY,EN

                  trafficking

                  vacuolar sorting protein 33A

                  1067

                  EN,LY

                  trafficking

                  valosin-containing protein

                  159

                  unknown

                  unclassified

                  vasodilator-stimulated phosphoprotein

                  503

                  PL

                  cell motility

                  vinculin

                  108

                  ME

                  cell motility

                  voltage-dependent anion channel 1

                  658

                  ER,LY,ME,NG,EX,PL,SY,MT

                  channel

                  voltage-dependent anion channel 2

                  657

                  SY,MT

                  channel

                  voltage-dependent anion channel 3

                  688

                  EN,ME,MT

                  channel

                  V-type proton ATPase subunit d 1

                  599

                  LY,EN,ME,SY

                  channel

                  WD repeat domain 1

                  304

                  EN,ME,EX

                  cell motility

                  Wiskott-Aldrich syndrome protein

                  323b

                  CY

                  cell motility

                  XRP2 protein

                  546

                  ME

                  signal trans.

                  zeta-chain associated protein kinase 70kDa

                  277

                  CY

                  signal trans.

                  397 individual proteins were identified to be associated with enriched secretory lysosomes from human T cell blasts. The proteins are listed by name, followed by individual spot numbers and the predicted/annotated subcellular localisation and function. Abbreviations: LY: lysosomes, ME: melanosomes, PL: platelet granules, SY: synaptosomes, EX: exosomes, CG: cytotoxic granules, NG: neuromelanin granules, EN: endosomes, MT: mitochondria, GO: Golgi, PE: peroxisomes, CY: cytoplasm, ER: endoplasmic reticulum and NU: nucleus. For detailed information on individual spots/proteins, please refer to the additional files.

                  Importantly, based on database annotations combining proteome analyses of different organelles [17], 70% of the 397 proteins were assigned to lysosomal or secretory vesicles (including cytolytic granules (CG), lysosomes (LY), exosomes (EX), endosomes (EN), melanosomes (ME), platelet granules (PL) and synaptosomes (SY)) (Table 1, Figure 5). The majority of the remaining 30% was classified as proteins of unknown (11%) or cytosolic (11%) localization, and as cytosolic or nuclear proteins (CY/NU, 3.5%). The low percentage of mitochondrial (MT, 1.5%), nuclear (NU, 0.8%), plasma membrane (PM, 0.3%) or endoplasmic strictly reticulum-associated (ER, 1.5%) and peroxisomal proteins (PE, 0.3%) again underscores the selective enrichment of lysosomal organelles in the present study. In terms of function, the classification revealed a large heterogeneity and a broad spectrum of potential activities. However, as expected, proteins associated with degradation, signal transduction, trafficking and immunity formed about 35% of the total proteome of enriched SL (Figure 5B). The important role of these organelles in cytotoxicity is also supported by the identified effector molecules perforin (#280) and granzyme A (#707, 717, 720, 724).
                  http://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_219_Fig5_HTML.jpg
                  Figure 5

                  Classification of the identified proteins according to their localization (A) and function (B). The cellular localization and function of 397 identified proteins were classified as detailed in material and methods. Lysosomal and secretory vesicles are represented by melanosomes (ME), lysosomes (LY), platelet granules (PL), endosomes (EN), synaptosomes (SY), exosomes (EX) or cytolytic granules (CG). Other cellular compartments are: cytosol (CY), nuclei (NU), peroxisomes (PE), plasma membrane (PM), mitochondria (MT), golgi (GO) or endoplasmic reticulum (ER).

                  Interestingly, and in contrast to the published SL proteome of NK cells [12], we did not detect significant amounts of granzyme B at the respective position in 2D gels from T cell blasts. However, this is in line with our previous observation that granzyme B might be stored in a separate compartment formed by electron dense granules that do not contain transmembrane FasL and that sediment as fraction 6 in our enrichment gradient [11]. To prove this result and address this issue in more detail, we started to analyze fraction 2 and fraction 6 vesicles (granzyme B granules). The direct comparison of the two granule populations by 2D DIGE and Western blotting clearly verified the result of the present analysis and provided first biochemical and proteomic evidence for two distinct species of cytotoxic effector vesicles in T cell blasts [18].

                  Surprisingly, it is still unknown to date whether functionally distinct TCRαβ and TCRγδ T cells, CD4+ and CD8+ T cells, vδ1+ and vδ2+ T cells, or normal and leukemic T cells also differ in terms of protein content and function of their lysosomal compartment(s). Based on the present description of the luminal proteome of FasL-containing secretory lysosomes in fully differentiated T cells, it will be possible to directly compare the content of cytotoxic effector organelles in different T cell subpopulations, e.g. by 2D difference gel electrophoresis. In addition, based on a larger set of marker proteins, the maturation of effector vesicles in the course of T cell activation can now be addressed in detail. Of note, using the applied protocol, we identified the luminal rather than the membrane proteome of this vesicular population. In addition, one has to consider that due to methodological limitations, the applied 2D technique might cover only about 20-30% of the total proteome and thus might be complemented in future studies employing LC-coupled mass spectrometric approaches.

                  Conclusion

                  We provide the first comprehensive proteome map of T cell-derived secretory lysosomes with only minor contaminations by cytosolic, nuclear or other proteins. This information will be useful to more precisely address the activation-dependent maturation and the specific distribution of effector organelles and proteins in individual T or NK cell populations in future studies.

                  Methods

                  Cells

                  Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coat preparations by Ficoll density gradient centrifugation. For the generation of PHA-stimulated lymphoblasts, T cells were purified by magnetic cell sorting (MACS) using cell isolation kits from Miltenyi Biotech (Bergisch Gladbach, Germany). The cells were stimulated with phytohemagglutinin A (PHA, 0.5 μg/ml, Remel, Lenexa, KS, USA) and expanded in the presence of irradiated EBV-transformed B cells and allogenic PBMC and subsequently with recombinant interleukin 2 (rIL-2, 100 U/ml, Chiron GmbH, Marburg, Germany). Before the cells were analyzed on day 12-14, dead cells were removed by Ficoll-gradient centrifugation resulting in a > 98% pure T cell population as judged by CD3 FACS analysis.

                  Confocal microscopy

                  Cells were fixed with 3% paraformaldehyde and permeabilized with 1% Triton X-100 as described [13]. The following antibodies were used: mouse IgG1 isotype-control MOPC-21 (Abcam, Cambridge, UK), anti-FasL mAb NOK1 (BD Biosciences, Heidelberg, Germany) with AlexaFluor488-conjugated goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany), anti-CD63 mAb clone MEM-259 (Immunotools, Friesoythe, Germany) conjugated to AlexaFluor555 (Invitrogen), anti-Granzyme A-FITC (Immunotools) and anti-Cathepsin B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with AlexaFluor488-conjugated donkey anti-goat IgG (Invitrogen). Stained samples were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and analyzed on a laser scanning microscope (LSM 510 Meta, Carl Zeiss, Jena, Germany) with appropriate filter settings. Images were acquired via scanning through the x-y-plane with 63 × objective lense. Laser intensity and detectors were adjusted to a uniformly negative signal of the control samples stained with control IgG and second step antibodies.

                  Subcellular fractionation

                  For subcellular fractionation and enrichment of secretory lysosomes, at least 4x108 T cells were used. The fractionation procedure has been recently described in detail [11]. Briefly, the cells were mechanically disrupted and organelles were enriched by differential centrifugation steps. The enriched organelles were then loaded on a discontinuous density gradient (4.4 ml volume) with 27%, 22.5%, 19%, 16%, 12%, 8% Optiprep® which is a 60% Iodixanol solution (Sigma, Deisenhofen, Germany) and subjected to ultracentrifugation. Interphases were collected from the top of the gradient resulting in six 400 μl fractions named and numbered 1 to 6. The protein content in each fraction was determined using a Coomassie Protein Assay Reagent (Thermo, Rockford, IL, USA).

                  Western blot analysis

                  For Western blotting, 5 μg of protein were separated by SDS-PAGE on pre-casted 4-12% gradient Bis-Tris gels (Invitrogen). After transfer to nitrocellulose (NC) membranes (Biometra, Goettingen, Germany) and blocking with 5% BSA or dry milk, the fractions were analyzed for subcellular marker proteins with the following antibodies: anti-FasL clone G-247.4 (BD Biosciences), anti-CD63 clone MEM-259 (Acris Antibodies, Herford, Germany), anti-LAMP-1 clone 25 (BD Biosciences), anti-cathepsin D clone CTD-19 (Sigma), anti-cytochrome oxidase IV (CoxIV) mAb clone 10G8D12C12 (1/1000, MitoScience, Eugene, OR, USA), anti pan-cadherin clone ab22744 (Abcam, Cambridge, UK) and horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (GE Healthcare, Munich, Germany). Membranes were prepared for reprobing by incubation in stripping solution (100 mM 2-mercaptoethanol, 2% SDS, 60 mM Tris) for 25 min at 56°C. ECL reagents in combination with Hyper Film (GE Healthcare) were used for chemiluminescence detection.

                  Transmission electron microscopy

                  Enriched organelles of fractions 2 and 5 were fixed with a mixture of 3% paraformaldehyde and 0.05% glutaraldehyde in PBS at 4°C overnight, washed in PBS, postfixed in 2% OsO4, dehydrated in ethanol, and embedded in araldite (Sigma, Deisenhofen, Germany). Ultrathin sections were mounted on formvar-coated grids and double-stained with a saturated solution of uranyl acetate in 70% methanol and lead citrate. The grids were examined with a Zeiss EM 900 transmission electron microscope equipped with a digital camera system.

                  2D electrophoresis, image analysis and spot picking

                  The 2D electrophoresis was performed as described before [11]. Briefly, SL pellets of fraction 2 were lysed on ice for 30 min with 30 μl lysis buffer (pH 8.5) containing 7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS. The supernatant was recovered after centrifugation for 20 min at 20.000 × g at 4°C. A total amount of 250 μg of protein was mixed with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% (v/v), IPG buffer pH 3-11 and 2% (w/v) DTT) and applied by cup-loading onto 24 cm non-linear pH 3-11 IPG gel strips for isoelectric focusing (IEF). The second dimension was performed on 26 × 20 cm large 12.5% polyacrylamide gels after reduction and alkylation using the Ettan DALTsix large vertical electrophoresis system from GE Healthcare. The gels were removed from the glass plates, stained with Flamingo Pink (Bio Rad), mounted on a non-backed gel frame, scanned on a Typhoon Trio imager (GE Healthcare) and analyzed using Image Master 6.0 (GE Healthcare). Selected spots were picked with a 2 mm picking head. The picked gels were again scanned to verify the correct location of the punched spots.

                  In-gel tryptic digestion and mass spectrometry

                  Gel plugs were washed with water and 12.5 mM ammonium bicarbonate (ABC) in 50% acetonitrile (ACN) and dehydrated in pure ACN. The dry gel pieces were rehydrated with 100 ng sequencing-grade trypsin (Serva, Heidelberg, Germany) in 5 mM ABC and tryptic in-gel digestion was performed at 37°C overnight. For peptide extraction, 0.3% trifluoroacetic acid (TFA) in ACN was added and the samples were sonicated for 15 min. The liquid phases were collected, lyophilized, redissolved in 0.5 to 1 μl MALDI matrix solution (3.2 mg/ml α-cyanohydroxycinnamic acid (Sigma) in 65% ACN/0.1% TFA), spotted onto 192-well stainless steel MALDI plates and air-dried. The samples were analyzed by peptide mass finger printing in positive reflectron mode followed by MSMS analyses of the most apparent five peptides using the 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems, Framingham, MA, USA) as described elsewhere [12]. Peptide mass spectra were processed by internal calibration with autolytic fragments of porcine trypsin with 25 ppm mass tolerance. MSMS spectra were acquired using default calibration updated prior to the run. Spectral data were searched against human proteins in the NCBI database (Homo sapiens, 192,176 entries) using MASCOT V2.0 (Matrix Sciences, London, UK).

                  Database analysis

                  Database searches with MASCOT were performed using the following parameters: the modification on cysteine residues by carbamidomethylation was set as obligate, methionine oxidation was considered as a potential modification; the maximum number of missed tryptic cleavages was one; the monoisotopic masses were considered and the mass tolerance was set to ± 50 ppm, and the fragment-ion mass tolerance was set to 0.2 Da (MS/MS). A protein was accepted to be identified when the total protein score reached or exceeded the MASCOT score threshold (≥ 65 with a probability of identification greater 95%). A repeated search against a randomized decoy database (http://​www.​matrixscience.​com/​help/​decoy_​help.​html) using the decoy.pl script and identical search parameters let to a false-positive rate of 1.2%.

                  The classification according the localization and function of individual proteins was based on the Uni-Prot knowledge base, the iHOP database [19] and the iProXpress database [17] available through the Protein Information Resource (PIR) (GUMC, Washington DC, USA). Identified proteins were searched in this organelle-proteome reference dataset according to their Uni-Prot numbers.

                  Declarations

                  Acknowledgements

                  This work was supported by grants from the German Research Foundation (SFB415 and 877), the Cluster of Excellence "Inflammation at Interfaces", the Innovationsfond Schleswig-Holstein, and the Medical Faculty of the Christian-Albrechts-University of Kiel.

                  Authors’ Affiliations

                  (1)
                  Institute of Immunology, Christian-Albrechts-University
                  (2)
                  Department of Zoophysiology, Zoological Institute, Christian-Albrechts-University
                  (3)
                  Institute of Anatomy, Christian-Albrechts-University

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                  © Schmidt et al; licensee BioMed Central Ltd. 2011

                  This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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