Table 1

Identified Phagosomal Proteins

MALDI-TOF-MS
ProteinAccession number*Prot ParamObservedRemarksPeptidesCoverageMS/MSTriton X-114Endo Phago
Mol WtpIMrpI
14-3-3P3521527771.14.73316004.2Involved in exocytosis through actin interaction.×
Acid ceramidaseQ9WV5444669.58.6840000NDLysosomal.13%××
A-X actinAAA3717041693.75.21MFCytoskeletal proteins.20%××
β-actinP0257041736.75.29410005.1Cytoskeletal proteins.21%××
γ-actinP0257141792.85.31405005.0Cytoskeletal proteins.41%×
AlixO8869596010.26.15946006.1Programmed cell death 6-interacting protein. Implicated in apoptosis, with ALG-2.7/713%
Annexin 5P4803635752.44.83346004.4Amount bound to phagosomes stays approximately the same as the phagosome matures.41%×
Apolipoprotein DP5191021529.74.8233000NDTransports a variety of ligands in a number of different contexts.××
ARP3P3239147371.15.61476005.4Actin-like protein 3. Cytoskeletal protein.9/1019%
Arylsulfatase BP5042931726.97.32413006.5Lysosomal.20%×
AshQ6305925206.35.89MF41%
ATP synth. βP1071956353.55.18479004.7Mitochondrial protein.14%××
CABP1Q6308147220.14.95498004.8Calcium-binding protein 1. Probable PDI P5 precursor.
CalnexinP3556467277.94.5120000NDRetains incorrectly folded glycoproteins in the ER.×××
CalreticulinP1421147994.54.33587003.8ER chaperone. Also found in T cell lytic granules.31%×
CAP 1P4012451574.87.16570007.0Adenylyl cyclase-associated protein 1. Located on the cell membrane.6/912%
CAPZ (α-actinin)P4775731345.45.47321005.3F-actin capping protein β subunit isoform 2.39%
Cathepsin AP1667553844.15.56MFLysosomal protective protein; carboxypeptidase C.10/1121%××
Cathepsin BP1060537279.85.57MFLysosomal cysteine protease.13/1832%×
Cathepsin DP1824244953.86.71MFLysosomal aspartic protease.26%××
Cathepsin LP0679737547.36.37MFLysosomal cysteine proteases.15%××
Cathepsin SO7037036909.66.16MFLysosomal cysteine proteases.29%×
Cathepsin ZQ9WUU733996.26.13MFLysosomal cysteine proteases.30%××
CoroninP3114651026.26.25MFShared homology with TACO.12/3220%
CyCAPO3564964055.54.91MFCyclophilin C-associated protein. Lysosomal.10/1320%×
cytochrome P450Q0676615681.86.71MFER membrane-bound protein.2/420%
EEA1Q14221162496.55.511266005.4Rab5 effector. Early endosome/phagosome.10%×
Elongation factor 1-a 1P1012650163.99.10447008.6EF-TU
EndoplasminP0811392475.74.741003004.3ER protein.13%
α-enolaseP1718247124.76.37MF2-phospho-d-glycerate hydro-lyase. Cytoplasmic.30%×
Epididymal secretory proteinQ9Z0J0164427.58233004.6Unknown function.×
ERP29P5255528574.86.23313005.9Found in the lumen of the ER.6/817%
Ferritin heavy chainP0952821066.65.53MFStores iron in a soluble, nontoxic,51%××
Ferritin light chain 1P2939120802.35.66MFreadily available form.8/848%×
FlotillinO0891747513.46.71427006.8Present in lipid rafts.35%
Galectin-3P1611027514.88.47322008.3MAC-2, laminin-binding protein. Galactose-specific lectin that binds IgE. Highest levels in activated macrophages. Involved in apoptosis.9/1035%×
GAPDHP16858358108.44341008.4Glycolysis.19%×
GILTQ9UL0829148.94.88MFIF-γ inducible lysosomal thiol reductase. May be involved in MHC class II–restricted antigen processing.×
GlucosylceramidaseP1743957621.87.6427000NDβ-glucocerebrosidase. Lysosomal. Membrane bound.9%××
β-glucuronidaseP1226574239.26.16MFLysosomal.16%××
GRB2P2935425206.35.89MFAssociates with tyrosine-phosphorylated proteins. Also interacts with Ras in the signaling pathway leading to DNA synthesis.25%
GRP 78P20029724225.07770004.6BIP. An ER chaperone.37%××
β-hexosaminidase αP29416605996.09MFN-acetyl-β-glucosaminidase, β-N-acetylhexosaminidase. Lysosomal.14%×
β-hexosaminidase βP2006061115.78.28MF19%×
HSC70TP1662770695.35.82MFHeat shock-related protein. Usually an ER or mitochondrial protein.
HSC71P0810970804.95.24MFHeat shock cognate protein30%×
HSP-60P1922660955.45.91MFMitochondrial matrix protein P1. Chaperonin. Interacts with P21RAS. Usually an ER or mitochondrial protein.6/927%×
HSP-70A45935708375.37MFCytoplasmic chaperone.27%×
HSP-70 precursorA4812773461.25.81MFCytoplasmic.31%
HSP-70 protein2P1715669740.85.58MF23%
HSP-73P08109708715.37709005.0Heat shock cognate 71 kD protein.24/3246%
HSP-90b (HSP-84)P11499831944.97900004.0Molecular chaperone. Has ATPase activity. Cytoplasmic. Interacts with the cytoskeleton as well.13%××
LactadherinP2195651465.26.02MFMilk fat globule-EGF factor 8 (MFG-E8). Antiviral activity.17%×
Lamin B1P1473366884.75.11MFComponent of the nuclear lamina.13/1325%
LAMP-1P1143843865.18.66135000NDLysosome-associated membrane glycoprotein 1. Type I membrane protein.×××
“LAMP-2, type B”P1704745647.07.05120000NDLysosome-associated membrane glycoprotein 2.×××
LegumainO8901749372.95.92376006Lysosomal cysteine endopeptidase.14%××
LIMP IIP2761553959.74.9195000NDLysosome membrane protein II. May act as a lysosomal receptor. Type II membrane protein. Lysosomal. Belongs to the CD36 family.18%××
Lysosomal acid lipase/cholesteryl ester hydrolaseP38571454156.4243000NDCrucial for the intracellular hydrolysis of cholesteryl esters and triglycerides. Lysosomal.15%×××
Lysosomal membrane glycoprotein-type BP1704643127.27.16120000NDVery similar to LAMP2 (P17047).×××
lysozyme C, type M (LYCM)P0890516688.99.1117000ND1,4-β-N-acetylmuraminidase C. Bacteriolytic. Enhances the activity of immunoagents.25%××
Macrophage capping proteinP2445239240.46.73MFActin-capping protein GCAP39; myc basic motif homolog-1.6/1017%
MHC class Ib, mature α chainCAA06194334195.0827000NDExpected to be secreted in soluble form due to absence of exon 5, which encodes the transmembrane domain.18%××
MPS1I5260373166.65.91MFMacrophage-specific protein. Upregulated during monocyte to macrophage differentiation.13%
Myosin heavy chain-AO8905518177.45.34155000NDNon muscle form.××
NapsinCAB8290745544.37.1355000NDMembrane-anchored aspartyl protease.××
NDK BQ01768173636.97211006.9Nucleotide diphosphate kinase B.61%
NSFP4646082565.46.52770006.4Vesicular-fusion protein. N-ethylmaleimide-sensitive factor.29%××
ORP150Q63617111289.15.111257004.8Oxygen regulated protein.
Palmitoyl-protein thioesteraseO8853134621.38.26MFRemoves thioester-like fatty acyl groups from modified cysteine residues in proteins or peptides during lysosomal degradation.23%××
PDI (ER-59)P0910357143.64.79555004.3Protein disulfide isomerase.6/1122%×
PDI (ER-60)P2777356621.35.99MFInvolved in MHC class I assembly.×
PGAM-BP2511328514.46.21315006.6Phosphoglycerate mutase, brain form.40%
ProhibitinP2414229820.15.57310005.2Present in lipid rafts.51%
RAB2P5399423547.56.0823000NDVesicular traffic. Associated with an intermediate compartment between the ER and Golgi apparatus.30%×
RAB3CQ6348224890.94.9825000NDProtein transport and vesicular traffic.14%×
RAB5CP352785496.14.8326500NDRegulates early endocytic/phagocytic trafficking.36%××
RAB7P5115023558.87.5324000NDRegulates late endocytic/phagocytic trafficking.44%××
RAB10O8838622540.98.5823200NDVesicular traffic and neurotransmitter release.30%×
RAB11BP4663824489.45.6425000NDInvolved in membrane recycling.17%××
RAB14P3528723926.95.8526500NDVesicular traffic and neurotransmitter release.40%×
RAP1BP0952620824.75.6522000NDInvolved in initiation of oxidative burst in neutrophils.39%××
RHO GDI αP1980323421.45.12275004.8RHO GDP-dissociation inhibitor 1×
RSP4P08865328544.79MF40S ribosomal protein SA. Cytoplasmic.18%×
SNAP-αP5492133194.95.16346005.0Soluble NSF attachment protein.76%×
SNAP-γQ9974734746.25.30374005.1Required to prepare intracellular membranes for fusion.30%×
StomatinP5411631403.36.46MFNDFound in lipid rafts, exposed on the cytoplasmic surface of the membrane.43%×
SynteninO8860132263.26.6633500NDLocalized in early endocytic compartments.×××
TCBP-49O7034137454.94.26472003.6Taipoxin-associated calcium binding protein 49.36%
TCP-1αP1198360448.65.82555005.7T-complex protein 1. Molecular chaperone. Known to play a role, in vitro, in the folding of actin and tubulin. Cytoplasmic.5/610%×
TCP-1βP8031457447.25.97555006.920%
TCP-1εP80316596245.72606005.618%
Thioreductase peroxidase 2P3570022176.58.26268008.4Cytoplasmic.4/420%
Ti-225 (ubiquitin C)Q6231714175.49.33142005.7Similar to human ubiquitin.42%
TMP21Q6358423276.86.0323200NDTransmembrane protein. Vesicular protein trafficking. Type I membrane protein. Present in Golgi cisternae.26%×
TRAILP5059233477.38.21313005.9TNF-related apoptosis inducing ligand.
Trimeric G α2P0875240470.95.28374004.9Guanine nucleotide-binding protein G(I)/G(S)/G(T). Adenylate cyclase-inhibiting Ga protein. Regulatory G-proteins of signaling cascades.13/1630%××
Trimeric G β1P0490137376.95.60359005.228%×
Trimeric G β2P54312373335.60357005.234%××
Tropomyosin 5P2110729020.64.75336004.2Cytoskeletal type.30%
Tubulin α-6P0521649909.34.96547004.8Microtubule proteins.14%
Tubulin β-5P0521849670.84.78495004.513/2438%×
UDPGTQ6455059662.78.7727000NDUDP-glucuronosyltransferase. E.R. protein.22%×
VAP33Q9QY7727271.57.6683000NDVesicle-associated membrane protein, associated protein A. Associated with ER and microtubules.40%×
v-ATPase α (catalytic subunit)P50516682685.62MFInvolved in phagosome acidification.44%×
v-ATPase βP5051756584.95.57MF9/1528%×
v-ATPase εP50518265889.28MF34%×
VDAC1Q6093230624.38.6328500NDVoltage-dependent anion-selective channel protein 1. Mitochondrial. Also found on the plasma membrane and endosomes.25%××
VimentinP2015253687.65.06MFClass III intermediate filaments.54%
AntitrypsinP3495546103.96.05MFSerum proteins adsorbed on latex beads during internalization. Highly enriched in our preparations. Endocytic cargo.18%
Apolipoprotein A-IP1549730276.35.71293005.0
BSAP0276969293.45.82MF16/2232%
α-S1 caseineP0266224528.94.98206004.335%
Hemoglobin αP0196615053.18.19163007.450%
Hemoglobin βP0208115859.26.51MF6/1146%
Putative gagP2309060511.77.63MFDuplan murine leukaemia virus.
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    Proteomic analysis of phagosome proteins. The 1- and 2-D gel methods combined allowed the identification of 116 phagosomal proteins directly from existing databases (including seven cargo molecules probably originating from the serum). In addition, 17 spots analyzed could not be matched to any entry in databases, while seven corresponded to ESTs (not shown), suggesting that they are novel proteins.

    *Whenever possible, the SWISS-PROT or TrEMBL accession numbers are listed. Otherwise, NCBI entries are used. MF, multiple forms. Observed pI values could not be evaluated for proteins identified by the 1-D gel method.

    MPS1 coverage is only NH2 terminal, and when this region is considered alone, the match percentage is very satisfactory; MALDI-TOF-MS, peptides, ratio of peptides that matched the expected mass of the theoretical trypsin digests. Coverage, percentage of the full-length sequence covered by the matching peptides; MS/MS, proteins further identified my MS/MS; Triton X-114, proteins that were identified from the detergent phase samples; Endo/phago, proteins previously reported to be present in endosomal/phagosomal compartments.