Analysis of Murine Natural Killer Cell Microsomal Proteins Using Two-Dimensional Liquid Chromatography Coupled to Tandem Electrospray Ionization Mass Spectrometry Josip Blonder,† Maria C. Rodriguez-Galan,‡ King C. Chan,† David A. Lucas,† Li-Rong Yu,† Thomas P. Conrads,† Haleem J. Issaq,† Howard A. Young,‡ and Timothy D. Veenstra*,† Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc. and Laboratory of Experimental Immunology, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201 Received April 5, 2004
This study describes the application of a single tube sample preparation technique coupled with multidimensional fractionation for the analysis of a complex membrane protein sample from murine natural killer (NK) cells. A solution-based method that facilitates the solubilization and tryptic digestion of integral membrane proteins is conjoined with strong cation exchange (SCX) liquid chromatography (LC) fractionation followed by microcapillary reversed-phase (µRP) LC tandem mass spectrometric analysis of each SCXLC fraction in second dimension. Sonication in buffered methanol solution was employed to solubilize, and tryptically digest murine NK cell microsomal proteins, allowing for the large-scale identification of integral membrane proteins, including the mapping of the membranespanning peptides. Bioinformatic analysis of the acquired tandem mass spectra versus the murine genome database resulted in 11 967 matching tryptic peptide sequences, corresponding to 5782 unique peptide identifications. These peptides resulted in identification of 2563 proteins of which 876 (34%) are classified as membrane proteins. Keywords: membrane proteins • large-scale membrane proteomics • solution-based multidimensional proteomic analysis
Introduction A large-scale analysis of complex protein mixtures is still an intricate task for membrane proteomics.1 The traditional global proteomic strategy, using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to analyze complex membrane protein mixtures, is restricted by its inability to solubilize and resolve this important class of proteins.2 Although a recent report showed noteworthy improvement in proteomic analysis of Saccharomyces cerevisiae membrane proteins using a gelbased approach, it required multiple 2D gel experiments along with several different sample preparations resulting in a more labor intensive sample handling process and less throughput than typically employed in standard 2D gel based approaches.3 Despite the improvement in the identification of insoluble membrane proteins, the limitation of analyzing very hydrophobic multipass integral membrane proteins still exists. Also, the sample quantity requirement exceeded the amounts readily obtainable from higher multicellular eucaryotic organisms (e.g., human or mouse).3 Hence, a significant trend has recently * To whom correspondence should be addressed. Dr. Timothy D. Veenstra, Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201. Phone: (301) 846-7296. Fax: (301) 846-6037. E-mail:
[email protected]. † Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc. ‡ Laboratory of Experimental Immunology, National Cancer Institute at Frederick.
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Published on Web 07/14/2004
developed toward gel-free membrane proteomics employing solution-based sample preparations that produce membrane proteome samples of diverse complexity ranging from whole cell lysates to well-characterized plasma membrane or lipid raft preparations.4-7 These membrane proteome samples are resolved using one-dimensional or different modes of multidimensional chromatographic separations typically ending with microcapillary reversed-phase liquid chromatography (µRPLC) coupled on-line to tandem mass spectrometry (MS/MS).1 Although the combination of separation techniques with the various sample preparation strategies used for global membrane proteomics is powerful and high throughput, each suffers from certain limitations such as incomplete solubilization of membrane proteins, inability to use specific enzyme proteolysis (e.g., trypsin) or the use of separation and ionization interfering compounds (e.g., detergents). Hence, there is a need for innovative strategies, which would enable more efficient sample preparation strategies of complex membrane protein mixtures that would parallel a large-scale proteomic analysis of cytosolic proteins. The objective of the present investigation is to utilize a solution-based multidimensional proteomic approach specifically optimized for large-scale qualitative analysis of complex protein mixtures containing membrane proteins with the aim to improve the overall capability to characterize murine natural killer (NK) cell integral membrane proteins. NK cells represent 10.1021/pr049927e CCC: $27.50
2004 American Chemical Society
Murine Natural Killer Cell Microsomal Proteins
research articles
a first line of defense of the innate immune system which recognizes and spontaneously kills aberrant cells that are infected by certain parasitic, bacterial, and viral pathogens or transformed by malignant growth.8 An analysis of the microsomal membrane proteome will facilitate an understanding of the critical roles of NK cell membrane proteins and concomitantly validate the utility of our multidimensional global proteomic strategy to identify key membrane proteins and receptors responsible for cytotoxic immune responses of murine NK cells.
Experimental Section Materials. Recombinant human (rh) IL-2 was obtained from Hoffmann-La Roche (Nutley, NJ), whereas RPMI 1640 media and ACK lysis buffer were purchased from BioWhittaker (Walkersville, MD). Fetal bovine serum (FBS) was purchased from Quality Biological Inc. (Gaithersburg, MD) while penicillin G sodium (100 U/ml), streptomycin sulfate (100 ug/mL) and L-glutamine (2mM) were obtained from Invitrogen-GIBCO (Carlsbad, CA). Sequencing grade-modified trypsin was obtained from Promega (Madison, WI). Deoxyribonuclease I, ammonium bicarbonate (NH4HCO3), iodoacetamide, EDTA, formic acid and phosphate-buffered saline (PBS) were obtained from Sigma (St. Louis, MO). HPLC grade methanol and acetonitrile were obtained from EM Science (Darmstadt, Germany). Bradford assay reagents and TCEP were obtained from Pierce (Rockford, IL). Ultrapure water was obtained using a Barnstead purification system (Dubuque, IA). Isolation of Natural Killer Cells. A proliferation of a large number of murine NK cells within liver and spleen was induced by i.v. administration of IL-2 cDNA as previously described.9 After injecting the C57BL/6 mice on day 0 with 5 µg of IL-2cDNA in 1.6 mL saline buffer, spleens were harvested on day 4 and single cell suspension obtained by passing the spleens through a metal mesh screen. Erythrocytes were lysed in ACK lysis buffer while mononuclears were washed and resuspended in 10% FBS RPMI 1640. The NK cell expansion is achieved by culturing mononuclear cells in ALAK media (RPMI 1640 supplemented with 10% FBS, 1 × 105 IU/mL of human IL-2, penicillin, streptomycin and L-glutamine)for 4-5 days.10 Phosphate buffered saline containing 1 mM EDTA was used to detach NK cells, which were washed in 3 mL of ice cold PBS and pelleted by 800 x g for 10 min at 4 °C and stored at -80 °C. Purity of NK cells was 70-80% as measured by NK1.1 and CD3 staining. The remaining 20-30% of the culture is likely comprised of NK toxic cells, T cells, B cells and macrophages. Preparation of Natural Killer Cell Microsomes. The pellet containing 40 × 109 NK cells was resuspended in 1 mL of 12.5 mM ammonium bicarbonate (NH4HCO3), pH 7.9, and kept on ice for 30 min followed by two cycles of snap freezing with liquid nitrogen and thawing in a water bath on 65 °C. The NH4HCO3 concentration was adjusted to 50 mM and 20 µg deoxyribonuclease I was added to the lysate which was further homogenized by 25 strokes in Dounce homogenizer. The homogenate was reduced using 15mM TCEP (Pierce, Rockford, IL) and alkylated using 30 mM iodoacetamide. The sample was further diluted with 50 mL of ice-cold 100 mM sodium carbonate, pH 11.3 and slowly agitated for 1 h at 4 °C. Microsomes were pelleted by ultracentrifugation at 100 000 × g for 1 h and the pellet was washed twice with double distilled water. The microsomal pellet was resolubilized in 10 mL of 25 mM ammonium bicarbonate and the same procedure was repeated twice and the final pellet was resolubilized in 500 µL
Figure 1. Sample preparation outline for multidimensional proteomic analysis of murine NK cell microsomal proteome. The first step involves cell disruption, followed by reduction, alkylation, pH carbonate treatment, and membrane isolation. Once the microsomes have been isolated using ultracentrifugation membrane proteins were solubilized using methanol facilitated extraction solubilization and direct tryptic proteolysis. The resulting digestate was fractionated using SCX chromatographic separation in the first dimension followed by microcapillary reversedphase LC-MS/MS of each SCX fraction.
of 25 mM NH4HCO3 followed by protein quantitation using the Bradford assay from Pierce (Rockford, IL). Microsomal proteins were extracted, solubilized, and proteolized as described elsewhere5. Briefly, methanol was added to 60% and microsomal proteins solubilized employing intermittent vortexing and sonication followed by direct trypsin (Promega, Madison, WI) digestion in the same tube using 1:20 enzyme/protein ratio. Strong Cation Exchange Fractionation. A strong cation exchange liquid chromatography (SCXLC) column (2.1 × 200 mm polysulfoethyl A, PolyLC Inc., (Columbia, MD) was employed to resolve the microsomal fraction digestate (∼200 µg) into 96 fractions using microcapillary liquid chromatography system Agilent Technologies Inc., (Palo Alto, CA). Peptide fractions were eluted by an ammonium formate/acetonitrile multistep gradient at a flow rate of 200 µL/min: 1% B/0-2 min, 10% B/62 min, 62% B/82 min, 100% B/85 min. Mobile phase A was 45% acetonitrile and mobile phase B was 45% acetonitrile containing 0.5 M ammonium formate (pH 3). The SCXLC fractions were lyophilized to dryness and reconstituted in 15 µL of 0.1% formic acid prior to µRPLC-MS/MS analysis. Microcapillary Reversed-Phase Liquid ChromatographyTandem Mass Spectrometry. Each of the 96 SCXLC fractions was analyzed by microcapillary reversed-phase liquid chromatography-tandem MS (µRPLC-MS/MS) using an Agilent 1100 capillary HPLC system (Agilent Technologies, Inc.) coupled to a LCQ Deca XP ion trap (IT)-MS (ThermoElectron, San Jose, CA). A reversed-phase microcapillary column (75 µm i.d. × 360 µm o.d. × 10 cm long) packed with 3 µm, 300 Å pore size, C-18 silica-bonded stationary phase (VYDAC, Torrence, CA) was used to fractionate the SCXLC fractions prior to MS/MS analysis. After injecting 5 µL of a sample onto the column, a 20 min wash with 98% buffer A (0.1% v/v formic acid) was applied and Journal of Proteome Research • Vol. 3, No. 4, 2004 863
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Figure 2. Gene ontology analysis of annotated proteins relative to their (A) subcellular localization, (B) biological process, and (C) molecular function.
peptides were eluted using a linear gradient of 2% solvent B (0.1% v/v formic acid in acetonitrile) to 85% solvent B in 120 min with a constant flow rate of 0.5 µL/min. The IT-MS was operated in a data-dependent mode where each full MS scan was followed by three consecutive MS/MS scans of each of the three most abundant peptide molecular ions that were dynamically selected for collision-induced dissociation (CID) using a normalized collision energy of 38%. The instrument was programmed to utilize an exclusion list so as to not perform redundant MS/MS of peptide molecular ions. The temperature of the heated capillary and electrospray voltage was 180 °C and 1.8 kV, respectively. 864
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Data Processing and Bioinformatic Analysis. The CID spectra detected from the µLC-MS/MS analysis were searched against the murine genome database using Turbo SEQUEST (ThermoElectron Inc.).11 Only those peptides identified as possessing fully tryptic termini (containing up to two missed internal trypsin cleavage sites) with cross-correlation scores (Xcorr) greater than 1.8 for singly charged peptides, 2.2 for doubly charged peptides and 3.3 for triply charged peptides [each with delta-correlation scores (∆Cn) greater than 0.1] were considered as legitimate identifications. The subcellular location of the identified proteins was elucidated from a gene ontology (GO) classification from the identified proteins (Expert Protein
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Murine Natural Killer Cell Microsomal Proteins
Table 1. Selected Subset of Hydrophobic Multipass Integral Membrane Proteins Identified from NK Cell Microsomal Fraction acc. no.a
Q99PR1 P36371 Q9WVD4 P17809 P32037 Q9WTK3 Q09143 Q9Z127 Q61168 Q9D8V0 P53986 P57787 P06795 P21447 P35379 O35874 O70579 Q9JJF9 Q9D2V8 Q9QZI9
protein description/peptide sequenceb
TMDc
GRAVYd
pepe
amino acid transporter Nat-2 K.LGEQVFGTTGK.L antigen peptide transporter 2 (TAP 2) R.HQAVLKEIQDAVAK.A chloride channel protein 5 K.WVADALGR.E facilitated glucose transporter, member 1 (GLUT-1) R.TFDEIASGFR.Q facilitated glucose transporter, member 3 (GLUT-3) K.KEEDQATEILQR.L GPI anchor attachment protein 1 K.YDLATVGK.A high-affinity cationic amino acid transporter-1 K.NLLGLGQQMLR.R L-type amino acids transporter 1 K.DMGQGDASNLQQK.L lysosomal-associated multitransmembrane protein K.HMNSAMEDSSSK.M minor histocompatibility antigen H13 K.SSSDMPETITSR.D monocarboxylate transporter 1 R.EGKEDEASTDVDEKPK.E monocarboxylate transporter 2 K.RPEVTEPEEVASEEK.L multidrug resistance protein 1 (MDR1) K.SGQTVALVGNSGC*GK.S multidrug resistance protein 3 (MDR3) K.SKDEIDNLDMSSK.D multidrug resistance protein(ATP-binding cassette) R.GYIQMTHLNK.T neutral amino acid transporter A (SATT) K.SEEETSPLVTHQNPAGPVAIAPELESK.E peroxisomal membrane protein PMP34 K.FRNEDIIPTNYK.G presenilin-like protein 2 (SPPL2A) K.DMKETLGDDITVK.M similar to tetracycline transporter-like protein R.FAAVTHSQDPPAEHR.L tumor differentially expressed protein 1 (TMS-1) K.DC*DVLVGFK.A
11
0.54989
2
5
0.68324
10
12
0.27975
2
12
0.53109
4
10
0.66490
5
7
0.40032
3
14
0.60418
2
9
0.71484
3
5
0.58773
2
7
0.32963
6
11
0.37208
7
12
0.59702
3
10
0.04811
3
11
0.02970
2
16
0.14103
5
9
0.51992
3
5
0.28792
5
9
0.57017
4
12
0.57039
5
11
0.51419
2
chgf
Xcorrg
2
2.5214
2
4.2567
2
2.7238
2
2.9383
2
4.5027
2
2.7253
2
2.7167
2
4.1267
2
3.1713
2
2.6451
2
4.7591
2
5.1203
2
3.8197
2
2.5291
2
2.8402
3
5.8285
2
2.8029
2
3.8132
2
2.6214
2
2.4557
a
Accession number, Swiss-Prot release of 08/22/03. b Sequence of the identified peptide with the highest Xcorr value. c Number of mapped transmembrane domains by TMHMM algorithm. d GRAVY value for each protein is calculated using the ProtParam algorithm. Proteins showing positive values were classified as hydrophobic. e Number of peptides identified for corresponding protein. f Charge state of precursor ion. g Cross-correlation score as determined by Sequest.
Analysis System proteomic server, http://www.expasy.org). The average hydropathy (GRAVY index) for selected proteins12 was calculated using the ProtParam software available at http:// us.expasy.org. Proteins exhibiting positive GRAVY values are considered hydrophobic and those with negative values are considered hydrophilic. The mapping of putative transmembrane domains of identified proteins was carried out using the transmembrane hidden Markov model (TMHMM) algorithm13 available at http://www.cbs.dtu.dk/services/TMHMM.
Results and Discussion The analysis resulted in the identification of 5782 unique peptides. These peptides could be directly mapped to 2563 unique proteins of which 876 (34%) were annotated as membrane or membrane associated proteins using gene ontology (GO) analysis (Figure 2). The same analysis showed that significant percentage (21%) of annotated proteins are involved in transport, cell communication, and response to external stimuli (Figure 2B), whereas Figure 2C shows that 40% of the proteins are involved in binding and transport activities based on their molecular functions. Mainly due to the hydrophobic nature of amino acids typically found within transmembrane domains it is possible, using bioinformatic analysis, to discriminate R-helical from non
R-helical membrane proteins and thereby enumerate their occurrence within the proteomic datasets. To determine the incidence of R-helical integral membrane proteins within our dataset the TMHMM algorithm was used, which is able to correctly predict 97-98% of present transmembrane helices and can discriminate between membrane and soluble proteins with specificity and sensitivity better than 99%.13 The analysis resulted in detection of 732 proteins that contain at least one transmembrane domain, of which 391 (53%) possessed 2-19 predicted transmembrane domains (Supplementary Table in the Supporting Information). These results indicate that 84% of 876 membrane proteins classified by GO analysis are also predicted as R-helical integral membrane proteins by TMHMM. The remaining 16% represent less hydrophobic integral membrane proteins (e.g., β-barrel membrane proteins) or soluble integral membrane proteins covalently attached to membrane bilayer (e.g., GPI-anchored membrane proteins). On the basis of the TMHMM algorithm analysis 29% of all proteins identified in this study contain at least one predicted transmembrane domain. This finding indicates that a 10-30% enrichment of membrane proteins above the current estimation that 20-25% of all open reading frames in currently sequenced genomes encode for R-helical membrane proteins.13 However, the actual enrichment may be higher since not all open reading frames Journal of Proteome Research • Vol. 3, No. 4, 2004 865
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Table 2. Selected Subset of Fully Tryptic Membrane Spanning Peptides Identified from Corresponding NK Cell Integral Membrane Proteins acc. no.a
P21958 O08734 P03911 P38378
Q99J27 Q8VDP6 Q9EPR4 Q99K84 Q9CZB0 Q9QZI8
protein description/transmembrane peptide sequence
TMDb
pepc
Antigen peptide transporter 1 (TAP1) R.VWVVGLSR.W R.WAILGLGVR.G Bcl-2 homologous antagonist/killer R.VVALLGFGYR.L NADH-ubiquinone oxidoreductase K.MPLYGVHLWLPK.A K.AHVEAPIAGSMILAAILLK.L Protein transport protein (Sec61 alpha-1) K.AFSPTTVNTGR.G R.GMEFEGAIIALFHLLATR.T R.ETSMVHELNR.Y Similar to acetyl-coenzyme A transporter K.LLWAPLVDAVYFK.N K.EHLALLAVPMVPLQIILPLLISK.Y Similar to phosphatidylinositol synthase R.MGLWVTAPIALLK.S K.SVISVIHLITAAR.N Solute carrier family 23, member 2 R.RVIQYGAALMLGLGMVGK.F Solute carrier family 29, member 1 R.REESGVPGPNSPPTNR.N R.EESGVPGPNSPPTNR.N Succinate dehydrogenase cytochrome 560 subunit K.SLC*LGPTLIYSAK.F K.FVLVFPLMYHSLNGIR.H Tumor differentially expressed protein 2 K.SQWTAVWVK.I
7
10
3
3
13
5
9
chgd
Xcorre
2 2
2.7038 2.7653
1
2.2810
2 2
3.6207 4.0474
2 3 1
2.2109 3.2331 2.6109
2 3
2.8613 3.8221
2 2
3.3434 3.7279
2
2.3699
2 2
3.1109 2.2072
2 2
2.3381 3.7751
2
2.4571
7
9
4
3
5
10
2
10
3
3
3
11
2
a
Accession number, Swiss-Prot Release of 08/22/03. b Number of mapped transmembrane domains by TMHMM algorithm. c Number of peptides identified for corresponding protein. d Charge state of precursor ion. e Cross-correlation score as determined by Sequest.
Table 3. Subset of Protein Receptors Identified within the NK Cell Microsomal Fraction acc. no.a
description
chg.b
Xcorrc
TMDd
pepe
Q61334 Q61335 P24668 P11835 P34902 Q09143 P09055 P11688 P20491 P11881 P16297 Q60652 Q60654 O88713 O55022 P27812 O54709 Q9R0A0 P30558 P47758 Q62186 Q8K328 Q8VDM0 P11942 Q62312 Q62351 P20334 Q9QZM4 P43406
B-cell receptor-associated protein (Bap29) B-cell receptor-associated protein (Bap31) CD MAN-6-P receptor) (CD-MPR) complement receptor (CD18) cytokine receptor (γ-C) (P64) ecotropic retrovirus receptor (ERR) fibronectin receptor (CD29 antigen) fibronectin receptor (CD49e) IgE Fc receptor γ-subunit (FcERI) inositol 1,4,5-trisphosphate receptor interleukin-2 receptor (CD122) killer cell lectin-like receptor (LY-49E) killer cell lectin-like receptor (LY-49G) MAFA killer cell lectin-like receptor G1 membrane progesterone receptor NK cell surface protein (NKR-P1) NKG2-D protein peroxisomal membrane receptor (PTS1) proteinase activated receptor (PAR-1) signal recognition particle receptor signal sequence receptor (SSR-δ) similar to complement receptor protein similar to lamin B receptor T-cell surface receptor (CD3) TGF-β type II receptor (TGFR-2) transferrin receptor protein (TfR1) tumor necrosis factor receptor tumor necrosis factor receptor (MK) vitronectin receptor (CD51 antigen)
2 2 1 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2
3.6761 4.8289 3.3997 5.5156 3.5673 2.7167 2.8523 3.5437 3.5670 2.3688 2.8994 4.5247 3.2331 2.9987 5.4556 3.4397 3.4412 2.9343 3.3504 4.5647 3.8195 3.3691 4.7025 2.5679 3.3600 3.7411 2.5186 3.8933 4.9428
3 2 1 1 1 14 1 1 2 6 2 1 1 1 1 1 1 1 7 1 1 1 7 1 1 1 1 2 1
5 5 3 9 3 2 2 2 2 2 4 3 2 3 2 3 3 3 2 7 5 2 4 2 3 5 4 2 9
a Accession number, Swiss-Prot Release of 08/22/03. b Charge state of precursor ion. c Cross-correlation score as determined by Sequest. d Number of mapped transmembrane domains by TMHMM algorithm. e Number of peptides identified for corresponding protein.
of the sequenced murine genome necessarily encode for all predicted membrane proteins under the given experimental conditions as predicted employing in-silico based calculations. 866
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The high-throughput nature of the present approach is exemplified by the ability to employ concomitant solubilization of membrane proteins along with direct and specific tryptic
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Murine Natural Killer Cell Microsomal Proteins
Table 4. Unbiased Identification of All Key Components of Class I and II MHC Antigen Processing and Presentation Pathway Including All Known Proteins of ER TAP1/TAP2 Peptide Loading Complex along with Selected Class I and II MHC Antigens acc. no.a
protein description/peptide sequence
TMDb
pepc
P21958
antigen peptide transporter 1 (TAP1) R.EGSVGEQGTHLQLMK.R R.KPLLLILDDATSALDAGNQLR.V antigen peptide transporter 2 (TAP2) R.HQAVLKEIQDAVAK.A R.DLEKDVYLVIR.R tapasin precursor (TAP-binding protein) R.FAYAPSALEGSPSLDAGPPPFGLEWR.R K.KPATLLLR.H calnexin precursor R.DEEEEEEKLEEK.Q R.VVDDWANDGWGLKK.A calreticulin precursor (ERp60) K.HEQNIDC*GGGYVK.L K.IKDPDAAKPEDWDER.A protein disulfide isomerase A3 (ERp57) K.ALEQFLQEYFDGNLK.R R.DLFSDGHSEFLK.A beta-2-microglobulin K.VEMSDMSFSK.D K.TPQIQVYSR.H class I MHC antigen H-2 (D-B R chain precursor) R.YISVGYVDNKEFVR.F R.FDSDAENPR.Y class I MHC antigen H-2 (K-B R chain precursor) R.YMEVGYVDDTEFVR.F R.YFVTAVSRPGLGEPR.Y class II MHC antigen H-2 (A β chain precursor) R.NGQEETVGVSSTQLIR.N R.RLEQPNVVISLSR.T
7
10
P36371 Q9R233 P35564 P14211 P27773 P01887 P01899 P01901 P14483
5 1 1 0 0 1 1 1 1
chgd
Xcorre
2 2
4.2407 3.5577
2 2
4.2567 3.8327
2 1
4.5734 1.9656
2 2
4.0751 3.8676
2 2
3.9185 3.6199
2 2
3.8560 3.5781
2 2
3.0743 2.6033
2 2
3.9311 3.8635
2 2
3.8640 3.2480
2 2
4.6833 3.6821
10 9 10 4 3 2 9 3 2
a Accession number, Swiss-Prot Release of 08/22/03. b Number of mapped transmembrane domains by TMHMM algorithm. c Number of peptides identified for corresponding protein. d Charge state of precursor ion. e Cross-correlation score as determined by Sequest.
Figure 3. Proteins of class I MHC antigen processing pathway unambiguously identified from microsomal proteome of murine NK cells. Endogenous proteins are proteolized in the proteasome complex, while degraded peptides are transported from cytoplasm to ER by TAP1/TAP2 ABC transporter. Within ER calnexin MHC I heavy chains chaperoned by calnexin uphold their structure while β2m macroglobulin is added by ERp57 soluble chaperon. Finally, loading complex is formed when calreticulin, class I MHC molecule and tapasin coalesce with TAP1/TAP2 heterodimer. Tapasin associates with TAP1/TAP2 complex and mediates interaction between class I MHC molecule with TAP1/TAP2. Stabilized MHC-peptide complex leaves ER through Golgi compartment. At the cell surface, MHCpeptide complex is presented to T lymphocytes for recognition.
proteolysis that generate peptides with optimal properties for both: electrospray ionization and MS/MS identification. The general applicability of the present approach to resolve hydrophobic, multipass integral membrane proteins from complex membrane protein mixture is exemplified in Table 1 that shows a subset of integral membrane proteins typically not amenable to discovery using a conventional 2D-PAGE based proteomic
investigation.2 In a recently published large-scale multidimensional proteomic analysis of a rat brain homogenate, Wu et al. succeeded in the identification of 1610 proteins, using nonspecific proteolysis of extramembranous portions of membrane proteins, of which 454 (28%) were predicted to be R-helical membrane proteins using TMHMM algorithm.7 In the present study, direct protein solubilization and specific enzymatic Journal of Proteome Research • Vol. 3, No. 4, 2004 867
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Table 5. Total of 15 Proteasome Complex Proteins Identified from Microsomal Proteome of Murine NK Cell along with Corresponding Peptide Sequences acc. no.a
Q8BK73 P97372 Q12920 Q9R1P4 Q9QUM9 O88685 P54775 P47210 Q92524 P14685 Q99JI4 P26516 Q9D8W5
protein description/peptide sequence
pepb
proteasome R.TTANAIYC*PPK.L proteasome activator complex subunit 2 (PA28β) R.AFYAELYHIISSNLEK.I proteasome activator complex subunit 3 (PA28γ) K.SNQQLVDIIEK.V proteasome subunit R type 1 R.NQYDNDVTVWSPQGR.I proteasome subunit R type 6 K.AINQGGLTSVAVR.G proteasome 26S protease regulatory subunit 6A K.LAGPQLVQMFIGDGAK.L proteasome 26S protease regulatory subunit 6B K.EFLHAQEEVKR.I proteasome 26S protease regulatory subunit 8 K.NIDINDVTPNC*R.V proteasome 26S protease regulatory subunit 10B K.VVSSSIVDKYIGESAR.L proteasome 26S non-ATPase regulatory subunit 3 R.VYEFLDKLDVVR.S proteasome 26S non-ATPase regulatory subunit 6 K.GAEILEVLHSLPAVR.Q proteasome 26S non-ATPase regulatory subunit 7 R.IVGWYHTGPK.L proteasome 26S non-ATPase regulatory subunit 12 R.LQEVIETLLSLEK.Q
3
chgc
Xcorrd
2
2.9456
2
5.1268
2
2.4966
2
3.7497
2
2.7389
2
3.8979
2
2.8092
2
2.6844
2
4.3096
2
3.2909
2
3.7268
2
2.4106
2
4.5729
3 3 3 1 2 1 3 2 4 1 1 1
a
Accession number, Swiss-Prot Release of 08/22/03. b Number of peptides identified for corresponding protein. c Charge state of precursor ion. d Crosscorrelation score as determined by Sequest.
Figure 4. Tandem mass spectra showing the unambiguous identification of two integral membrane peptides (VWVVGLSR and WAILGLGYR) originating from antigen peptide transporter-1 (TAP1). A total of 10 unique peptides from this protein were identified by MS/MS (italicized). The transmembrane spanning domains of TAP1 are shown in bold font. The y-axis represents the relative intensity of each product ion.
proteolysis was applied to NK cell microsomal fraction. The total number of 2563 identified proteins along with 732 predicted integral membrane proteins reported in this study is significantly higher than that reported by Wu et al., (1610 868
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and 454 integral membrane proteins).7 Since each study was carried out on different samples and employed dissimilar sample preparations in terms of protein isolation, solubilization and enzymatic digestion, the present comparison indicate that
research articles
Murine Natural Killer Cell Microsomal Proteins
each approach allowed for wide-ranging analysis of complex protein mixtures containing insoluble integral membrane proteins. The capability of a “single tube” technique to solubilize membrane proteins implies that the identification of membrane spanning peptides from a complex protein mixture should also be expected.5 Indeed, Table 2 shows a selected subset of integral membrane proteins that were identified by membrane spanning peptides as mapped using TMHMM algorithm. These results show that the protein solubilization technique used in this study enabled the accessibility of trypsin cleavage sites localized within the membrane bilayer. Obviously, the ability of the presented method to identify these peptides depends not only on solubilization efficacy but also on the presence of arginyl or lysyl residues within or in the close proximity to membrane domains and also on presence or absence of post-translational protein modifications which are very common among membrane proteins.1 The utility of a global proteomic analysis can also be measured by the ability to identify specific membrane proteins known to be exclusively expressed by a given organism, organ or cell type. Table 3 shows a selected subset of NK cell-specific receptors identified including NK cell surface proteins as Ly49a, Ly49g which are among the best understood inhibitory receptors expressed specifically by murine NK cells. Also identified in this study were the activating receptors NKG2D and MAFA that are expressed by mouse and human NK-cells.8 Global proteomic profiles of murine NK cell integral membrane proteins are not yet available, mainly because standard 2D-PAGE-based approaches have proven inadequate for analyzing membrane proteomes. Consequently, the effectiveness of solution-based multidimensional membrane proteomic strategy is critical since it is well accepted that any given onedimensional chromatographic or electrophoretic separation is not sufficient for comprehensive investigations of complex integral membrane protein mixtures. In this study, key components of TAP1/TAP2 peptide loading complex shown in Table 4 and Figure 3 were identified, resulting in a comprehensive mapping of the class I antigen processing pathway.15 Since the NK cell population used in this study was 70-80% pure as determined by NK1.1 and CD3 staining (data not shown) it is possible that some of the protein identifications arose from the contaminating cell population including NK T-cells. Some of the proteins that are expected to originate from the contaminating cell population include B-cell receptor associated protein (Bap)29, Bap31, IgE Fc receptor γ-subunit, T-cell surface receptor, and Class II MHC antigen H-2. Thus, the specific association of proteins identified in this study with NK cells awaits analysis of highly purified NK cell population. All proteins shown in Figure 3 were identified by multiple peptides (Table 4) as well as a significant number of proteins from proteasome complex (Table 5) indicating all-inclusive proteomic plotting of MHC class I antigen-presenting pathway. The single proteomic investigation of microsomal proteome described in this work was able to positively identify all key proteins of an important immune response modulating system, previously elucidated through numerous biological and biochemical studies. An important aspect of the membrane proteome analytical technique presented in this manuscript is its capability of analyzing transmembrane domains along with those peptides arising from soluble luminal or cytoplasmic domains. The MS/ MS spectra shown in Figure 4 indicate the unambiguous
identification of two peptides that completely span the second transmembrane domain of the pore protein domain of TAP1, which was also identified from the MS/MS spectra of 8 additional unique peptides shown in Table 4 and supplementary data.16 TAP1 is part of the heterodimeric ABC peptide transporter TAP1/TAP2 that translocates peptides from the cytosol to the lumen of the endoplasmatic reticulum for binding to MHC class I molecules. The TAP1/TAP2 transporter is a component of the peptide loading complex that is made of β2-microglobulin, chaperones calreticulin (ERp60), protein disulfide isomerase (ERp57) and tapasin.17 The results presented validate the utility and high throughput performance of proteomic strategy described in this work indicating its ability to probe membrane protein complexes regardless of the solubility of each constitutive protein.
Conclusion In this study, a solution-based method developed for the analysis of membrane proteins5 has been used to characterize proteins within the microsomal fraction of murine NK cells. This approach relies on efficient solubilization of integral membrane proteins and their direct tryptic digestion in a “single tube” preparation procedure. On the basis of the results presented, the methodology described in this work integrates directly into conventional proteomic investigations that employ multidimensional separations and mass spectrometry for global protein identification. Unlike other methods for global membrane proteomics the present approach does not require the use of detergents, strong organic acids, harsh chemical digestion protocols, or nonspecific proteolysis. Hence, the simplicity of this single tube procedure used for solubilization, and direct tryptic digestion is comparable to the global proteomic strategies employed for the large-scale proteomic analysis of soluble proteins. Additionally, the overall protein preparation presented in this work is not limited to the recovery of only the extramembranous portions of membrane proteins, but allows the interrogation of membrane spanning domains which is of significant benefit for characterization of multipass integral membrane proteins that possess less extensive extramembranous protein domains
Acknowledgment. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1CO-12400. Supporting Information Available: A comprehensive list of identified proteins and peptides from NK cells microsomal fraction (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Wu, C. C.; Yates, J. R. Nat. Biotechnol. 2003, 21, 262-267. (2) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054-1070. (3) Pedersen, S. K.; Harry, J. L.; Sebastian, L.; Baker, J.; Traini, M. D.; McCarthy, J. T.; Manoharan, A.; Wilkins, M. R.; Gooley, A. A.; Righetti, P. G.; Packer, N. H.; Williams, K. L.; Herbert, B. R. J. Proteome Res. 2003, 2, 303-311. (4) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351360. (5) Blonder, J.; Conrads, T. P.; Yu, L. R.; Terumuma, A.; Janini, G. M.; Issaq, H. J.; Vogel, J.; Veenstra, T. D. Proteomics 2004, 4, 3145. (6) Foster, L. J.; de Hoog, C. L.; Mann, M. Proc. Natl. Acad. Sci. U.S.A 2003, 100, 5813-5818.
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research articles (7) Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R. Nat. Biotechnol. 2003, 21, 532-538. (8) Yokoyama, W. M.; Plougastel, B. F. M. Nat. Rev. Immunol. 2003, 3, 304-316. (9) Fogler, W. E.; Volker, K.; McCormick, K. L.; Watanabe, M.; Ortaldo, J. R.; Wiltrout, R. H. J. Immunol. 1996, 156, 4707-4714. (10) Ortaldo, J. R.; Winkler-Pickett, R.; Willette-Brown, J.; Wange, R. L.; Anderson, S. K.; Palumbo, G. J.; Mason, L. H.; McVicar, D. W. J. Immunol. 1999, 163, 5269-5277. (11) Eng, J. K.; McCormack, A. L.; Yates, J. R. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989.
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Blonder et al. (12) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105-132. (13) Krogh, A.; Larsson, B.; von Heijne, G.; Sonnhammer, E. L. J. Mol. Biol. 2001, 305, 567-580. (14) Walter, P.; Blobel, G. Methods Enzymol. 1983, 96, 84-93. (15) Abele, R.; Tampe, R. Biochim. Biophys. Acta 1999, 1461, 405419. (16) Reits, E. A.; Griekspoor, A. C.; Neefjes, J. Immunol. Today 2000, 21, 598-600. (17) Tan, P.; Kropshofer, H.; Mandelboim, O.; Bulbuc, N.; Hammerling, G. J.; Momburg, F. J. Immunol. 2002, 168, 1950-1960.
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