Open Access

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

  • Hendrik Schmidt1,
  • Christoph Gelhaus2,
  • Melanie Nebendahl1,
  • Marcus Lettau1,
  • Ralph Lucius3,
  • Matthias Leippe2,
  • Dietrich Kabelitz1 and
  • Ottmar Janssen1Email author
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].
https://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_Article_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.
https://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_Article_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.
https://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_Article_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.
https://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_Article_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).
https://static-content.springer.com/image/art%3A10.1186%2F1478-811X-9-4/MediaObjects/12964_2010_Article_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 article is published under license to BioMed Central Ltd. 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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