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• W2103064512 abstract "Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers. Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers. The lysosome is an acidic, membrane-delimited organelle that is responsible for the degradation and recycling of macromolecules, playing a role in endocytosis and autophagy (1Holtzman E. Lysosomes. Plenum Press, New York1989Crossref Google Scholar). Most of the resident hydrolases (e.g. proteases, glycosidases, lipases, phosphatases, sulfatases, and nucleases) and accessory proteins (e.g. activator proteins) are transported to the lysosome by the mannose 6-phosphate (Man-6-P) 1The abbreviations used are: Man-6-P, mannose 6-phosphate; MPR, Man-6-P receptor; CI-MPR, cation-independent MPR; sCI-MPR, soluble cation-independent MPR; HS, Heremans-Schmid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; 1D, one-dimensional; 2D, two-dimensional; GPM, Global Proteome Machine; BTD, biotinidase; PTGDS, prostaglandin-H2 d-isomerase; CP, ceruloplasmin; CPVL, carboxypeptidase vitellogenic-like; SMPDL3A, acid sphingomyelinase-like protein 3a; CREG1, cellular repressor of E1A-regulated gene transcription; EPDR1, mammalian ependymin-related protein. 1The abbreviations used are: Man-6-P, mannose 6-phosphate; MPR, Man-6-P receptor; CI-MPR, cation-independent MPR; sCI-MPR, soluble cation-independent MPR; HS, Heremans-Schmid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; 1D, one-dimensional; 2D, two-dimensional; GPM, Global Proteome Machine; BTD, biotinidase; PTGDS, prostaglandin-H2 d-isomerase; CP, ceruloplasmin; CPVL, carboxypeptidase vitellogenic-like; SMPDL3A, acid sphingomyelinase-like protein 3a; CREG1, cellular repressor of E1A-regulated gene transcription; EPDR1, mammalian ependymin-related protein. targeting pathway. Like many other glycoproteins, soluble lysosomal proteins are synthesized in the endoplasmic reticulum and are cotranslationally glycosylated on select asparagine residues. As these proteins move through the secretory pathway, the lysosomal proteins are selectively recognized by a phosphotransferase that initiates a two-step reaction that results in the generation of the Man-6-P modification on specific N-linked oligosaccharides. The modified proteins are then recognized by two Man-6-P receptors (MPRs), the cation-dependent MPR and the cation-independent (CI-) MPR (2Ghosh P. Dahms N.M. Kornfeld S. Mannose 6-phosphate receptors: new twists in the tale.Nat. Rev. Mol. Cell. Biol. 2003; 4: 202-212Crossref PubMed Scopus (798) Google Scholar). These receptors bind the phosphorylated lysosomal proteins in the neutral environment of the trans-Golgi network and travel to an acidic prelysosomal compartment in which the low pH promotes dissociation of the receptors and ligands. The receptors then recycle back to the Golgi to repeat the process or to the plasma membrane. Here the CI-MPR can function in the endocytosis and lysosomal targeting of extracellular Man-6-P glycoproteins. The primary location and site of function of lysosomal enzymes is by definition intracellular. However, lysosomal activities have also been identified in a variety of extracellular environments including plasma (3Lentner C. Lentner C. Wink A. Geigy Scientific Tables. Ciba-Geigy, West Caldwell, NJ1984: 166-199Google Scholar). These proteins may be delivered to the plasma through leakage from dead or dying cells, through mobilization of secretory lysosome or granule contents, or by the release of lysosomal residual bodies by cell defecation. It is also possible that a portion of the newly synthesized lysosomal proteins escape the intracellular targeting pathway for transport to the lysosome and are secreted. Evidence for the latter is well documented; for example, in cultured mouse embryonic fibroblasts, wild-type cells secrete a small proportion of Man-6-P glycoproteins (∼10% of that secreted in the absence of both MPRs, depending on the protein) (4Ludwig T. Munier-Lehmann H. Bauer U. Hollinshead M. Ovitt C. Lobel P. Hoflack B. Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts.EMBO J. 1994; 13: 3430-3437Crossref PubMed Scopus (133) Google Scholar, 5Pohlmann R. Boeker M.W. von Figura K. The two mannose 6-phosphate receptors transport distinct complements of lysosomal proteins.J. Biol. Chem. 1995; 270: 27311-27318Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 6Kasper D. Dittmer F. von Figura K. Pohlmann R. Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes.J. Cell Biol. 1996; 134: 615-623Crossref PubMed Scopus (103) Google Scholar, 7Munier-Lehmann H. Mauxion F. Bauer U. Lobel P. Hoflack B. Re-expression of the mannose 6-phosphate receptors in receptor-deficient fibroblasts. Complementary function of the two mannose 6-phosphate receptors in lysosomal enzyme targeting.J. Biol. Chem. 1996; 271: 15166-15174Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), indicating that the sorting of newly synthesized lysosomal proteins is not absolutely efficient. The biological significance of circulating lysosomal proteins is unclear, but there are several possibilities. First, some hydrolases may be synthesized and released by one cell type and delivered to another, and so circulating lysosomal proteins may represent intermediates in this process. Second, circulating lysosomal proteins might have a specific function in plasma. Third, the presence of lysosomal proteins in plasma may simply represent the steady state levels reflecting the balance between the rate of unwanted appearance in plasma (due to leakage and/or lack of absolute fidelity in the intracellular targeting system) versus the rate of uptake and clearance. Regardless of biological function, circulating lysosomal hydrolases potentially represent valuable biomarkers for the study and diagnosis of human diseases. Mutations in genes encoding lysosomal proteins result in over 40 storage diseases (for a review, see Ref. 8Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. 8th Ed. The Metabolic & Molecular Bases of Inherited Disease. III. McGraw-Hill, New York2001: 134-3894Google Scholar). In addition, lysosomal activities have also been indirectly implicated in more widespread pathogenic processes (9Nixon R.A. Cataldo A.M. Mathews P.M. The endosomal-lysosomal system of neurons in Alzheimer’s disease pathogenesis: a review.Neurochem. Res. 2000; 25: 1161-1172Crossref PubMed Scopus (280) Google Scholar, 10Bromme D. Kaleta J. Thiol-dependent cathepsins: pathophysiological implications and recent advances in inhibitor design.Curr. Pharm. Des. 2002; 8: 1639-1658Crossref PubMed Scopus (93) Google Scholar, 11Du H. Grabowski G.A. Lysosomal acid lipase and atherosclerosis.Curr. Opin. Lipidol. 2004; 15: 539-544Crossref PubMed Scopus (33) Google Scholar, 12Fehrenbacher N. Jaattela M. Lysosomes as targets for cancer therapy.Cancer Res. 2005; 65: 2993-2995Crossref PubMed Scopus (278) Google Scholar) including tumor invasion and metastasis, Alzheimer disease, rheumatoid arthritis, and atherosclerosis as well as in normal processes such as aging (13Lynch G. Bi X. Lysosomes and brain aging in mammals.Neurochem. Res. 2003; 28: 1725-1734Crossref PubMed Scopus (36) Google Scholar) and immune system function (14Stinchcombe J. Bossi G. Griffiths G.M. Linking albinism and immunity: the secrets of secretory lysosomes.Science. 2004; 305: 55-59Crossref PubMed Scopus (306) Google Scholar). As a class, lysosomal proteins represent a relatively small subgroup of the plasma proteome that should be amenable to quantitative analysis. In this study, we set out to characterize the human plasma proteome of mannose 6-phosphate glycoproteins to provide a reference dataset as a diagnostic tool for lysosomal storage diseases that could also facilitate further studies of the role of these proteins in other human conditions. We also viewed plasma as a promising source for the identification of potentially novel lysosomal proteins in our ongoing effort to identify components of the lysosomal proteome (15Sleat D.E. Sohar I. Lackland H. Majercak J. Lobel P. Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis.J. Biol. Chem. 1996; 271: 19191-19198Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16Sleat D.E. Kraus S.R. Sohar I. Lackland H. Lobel P. α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine.Biochem. J. 1997; 324: 33-39Crossref PubMed Scopus (17) Google Scholar, 17Sleat D.E. Lackland H. Wang Y. Sohar I. Xiao G. Li H. Lobel P. The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.Proteomics. 2005; 5: 1520-1532Crossref PubMed Scopus (78) Google Scholar). Given the complexity of the plasma protein population and the wide range of protein abundances, it is not currently feasible to directly detect lysosomal proteins in unfractionated plasma using proteomic methods due to their extremely low abundance compared with classical plasma proteins (18Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol. Cell. Proteomics. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3557) Google Scholar). We have therefore used an approach in which the Man-6-P-containing lysosomal proteins are purified by affinity chromatography on immobilized MPR (15Sleat D.E. Sohar I. Lackland H. Majercak J. Lobel P. Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis.J. Biol. Chem. 1996; 271: 19191-19198Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16Sleat D.E. Kraus S.R. Sohar I. Lackland H. Lobel P. α-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine.Biochem. J. 1997; 324: 33-39Crossref PubMed Scopus (17) Google Scholar, 17Sleat D.E. Lackland H. Wang Y. Sohar I. Xiao G. Li H. Lobel P. The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.Proteomics. 2005; 5: 1520-1532Crossref PubMed Scopus (78) Google Scholar, 19Journet A. Chapel A. Kieffer S. Louwagie M. Luche S. Garin J. Towards a human repertoire of monocytic lysosomal proteins.Electrophoresis. 2000; 21: 3411-3419Crossref PubMed Scopus (65) Google Scholar, 20Journet A. Chapel A. Kieffer S. Roux F. Garin J. Proteomic analysis of human lysosomes: application to monocytic and breast cancer cells.Proteomics. 2002; 2: 1026-1040Crossref PubMed Scopus (90) Google Scholar, 21Kollmann K. Mutenda K.E. Balleininger M. Eckermann E. von Figura K. Schmidt B. Lubke T. Identification of novel lysosomal matrix proteins by proteome analysis.Proteomics. 2005; 5: 3966-3978Crossref PubMed Scopus (74) Google Scholar, 22Czupalla C. Mansukoski H. Riedl T. Thiel D. Krause E. Hoflack B. Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism.Mol. Cell. Proteomics. 2006; 5: 134-143Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). As expected, we identified many lysosomal proteins, but we also purified many proteins that have not previously been assigned either Man-6-P or lysosomal function. Many of these proteins are highly abundant plasma glycoproteins that we initially suspected to represent likely contaminants that do not contain Man-6-P. Therefore, to distinguish between true Man-6-P glycoproteins and contaminants, we used a technique to directly identify Man-6-P-modified glycopeptides. Unexpectedly we found that a small proportion of multiple abundant classical plasma proteins exist as Man-6-P-containing glycoforms. Refrozen lots of thawed human plasma were generously provided by V. I. Technologies, Inc. (Melville, NY). Plasma Man-6-P glycoproteins were purified using a modification of the affinity protocol described for human brain (17Sleat D.E. Lackland H. Wang Y. Sohar I. Xiao G. Li H. Lobel P. The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.Proteomics. 2005; 5: 1520-1532Crossref PubMed Scopus (78) Google Scholar). For each purification, ∼2 liters of frozen human plasma were rapidly thawed at 37 °C and then clarified by centrifugation at 13,000 × g for 90 min at 4 °C. The supernatant was filtered through six layers of cheesecloth and then added to an equal volume of ice-cold PBS containing 10 mm β-glycerophosphate, 10 mm EDTA, 2% Triton X-100, 0.4% Tween 20, and 0.2 mm Pefabloc (Pentafarm, Basel, Switzerland). The diluted plasma was then applied to a column of immobilized pentamannosyl phosphate-aminoethyl agarose (100-ml bed volume) to remove circulating soluble cation-independent MPR (sCI-MPR), and the flow-through was applied to a column of immobilized sCI-MPR (100-ml bed volume at a coupling density of 3.3 mg/ml) at a flow rate of ∼200 ml/h to capture circulating Man-6-P glycoproteins. The MPR resin was batch washed with 3 × 1 column volume of PBS containing 5 mm EDTA, 5 mm β-glycerophosphate, 1% Triton X-100 and then with 3 × 1 column volume of the same buffer without Triton X-100. The column was flow-washed with 20 column volumes of PBS/EDTA/β-glycerophosphate at 125 ml/h and then eluted with 2 column volumes of the same buffer containing 10 mm Man-6-P at a flow rate of 100 ml/h. Fractions containing Man-6-P glycoproteins were identified by protein and β-mannosidase assay, pooled, concentrated by ultrafiltration (Centriprep-10, Millipore, Billerica, MA), and buffer-exchanged to either 100 mm ammonium bicarbonate (single purification protocol) or PBS/EDTA/β-glycerophosphate (first elution from the double purification protocol). For repurification, the buffer-exchanged Man-6-P eluate was reapplied to the column of immobilized MPR and purified essentially as described above. The Man-6-P eluate was buffer-exchanged with 100 mm ammonium bicarbonate. Purified proteins were stored at −80 °C, and volatile salts were removed by lyophilization prior to subsequent analyses. First dimension isoelectric focusing was conducted using pH 3–7 or 3–10 non-linear Immobiline DryStrips (Amersham Biosciences), and second dimension gel electrophoresis was conducted using bis-Tris·HCl-buffered (pH 6.4) 10% Duracryl (Genomic Solutions, Ann Arbor, MI) gels run with MOPS running buffer. Transfer to nitrocellulose and detection of Man-6-P glycoproteins using iodinated sCI-MPR were as described previously (15Sleat D.E. Sohar I. Lackland H. Majercak J. Lobel P. Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis.J. Biol. Chem. 1996; 271: 19191-19198Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 22Czupalla C. Mansukoski H. Riedl T. Thiel D. Krause E. Hoflack B. Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism.Mol. Cell. Proteomics. 2006; 5: 134-143Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Fractionated proteins were visualized using either a colloidal Coomassie Blue staining protocol (23Valenzano K.J. Kallay L.M. Lobel P. An assay to detect glycoproteins that contain mannose 6-phosphate.Anal. Biochem. 1993; 209: 156-162Crossref PubMed Scopus (42) Google Scholar) or SYPRO Ruby (Invitrogen). Proteolytic digests of MPR-affinity purified plasma proteins were analyzed by nanospray LC-MS/MS using a ThermoElectron LTQ linear ion trap (ThermoElectron, San Jose, CA) and a Waters Micromass Q-TOF API US (Waters, Milford, MA) mass spectrometer. For both the singly and doubly purified human plasma samples, MPR-affinity purified mixtures were digested with trypsin in solution prior to LC-MS/MS. Forty micrograms of each sample were also fractionated by 1D SDS-PAGE (Novex 10% NuPage gels with MOPS buffer (Invitrogen)), and ∼30 gel slices were digested with trypsin. For the doubly purified sample alone, 40 μg of purified proteins were fractionated by two-dimensional (2D) gel electrophoresis, and ∼60 spots were excised and digested with trypsin. The LTQ was used to analyze digests of all the samples discussed above. Solution digests of each of the singly and doubly purified samples were analyzed in duplicate. Instrument, chromatographic conditions, and generation of peak lists were as described previously (24Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. Carnemolla B. Orecchia P. Zardi L. Righetti P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1594) Google Scholar) except for the analysis of gel slices, which was conducted using a 50-min gradient. For the Q-TOF, tryptic digests of each MPR-affinity purified sample (5 μg) or tryptic digests of 1D gel slices were fractionated using a Micromass CapLC. Peptides were first desalted on a 300-μm × 1-mm PepMap C18 trap column with 0.1% formic acid in HPLC grade water at a flow rate of 20 μl/min. After desalting for 3 min, peptides were back-flushed onto an LC Packings 75-μm × 15-cm C18 nanocolumn (3 μm, 100 Å) at a flow rate of 200 nl/min. For solution digests of MPR-affinity purified proteins, peptides were eluted on a 170-min gradient of 3–42% acetonitrile in 0.1% formic acid. For digests of 1D gel slices, peptides were eluted on a 30-min gradient of 3–42% acetonitrile in 0.1% formic acid. The scan time for MS and MS/MS were 1.5 and 2.0 s, respectively. Mass ranges for the MS survey scan and MS/MS were m/z 300–1900 and m/z 50–1900, respectively. The top three multiply charged ions with an MS peak intensity greater than 30 counts/scan were chosen for MS/MS fragmentation with a precursor ion dynamic exclusion of 60 s. Instrument switching from MS/MS to MS mode occurred when either total MS/MS ion counts were over 5000 or when the total MS/MS scan time was over 6.0 s. Raw data were processed by ProteinLynx 2.0 to generate PKL files without peak threshold intensity limits (fast deisotoping) and with automatic charge state determination. Peptide assignment and protein identification were conducted using a local implementation of X!Tandem Version 2005.12.01 (GPM-USB, Beavis Informatics Ltd., Winnipeg, Canada) to search the human proteome database (the February 2006 assembly of the National Center for Biotechnology Information (NCBI) 35, which contains a total of 33,869 entries representing 30,352 unique sequences) as described previously (24Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. Carnemolla B. Orecchia P. Zardi L. Righetti P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1594) Google Scholar). Database searching using data generated with the Q-TOF was identical except that the parent ion mass error was ±0.5 Da. Methods for analysis of LC-MS/MS data from proteolytic digests of unfractionated mixtures or gel slices and for deglycosylated Man-6-P glycopeptides (see below) have been described previously (24Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. Carnemolla B. Orecchia P. Zardi L. Righetti P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1594) Google Scholar). Data were exported into Microsoft Excel spreadsheets and filtered for threshold significance as described below. To avoid redundancy arising from proteins present in the human proteome database under multiple names and/or accession numbers, a list of ENSP numbers was compiled with all numbers corresponding to a single protein referenced to a single primary number. Protein assignments were compared with this database to ensure that multiple accession numbers representing the same protein were not listed. The presence of Man-6-P on MPR-purified proteins was verified using a recently developed protocol for the purification and identification of glycopeptides containing Man-6-P from an unfractionated proteolytic digest of MPR-purified proteins (24Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. Carnemolla B. Orecchia P. Zardi L. Righetti P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1594) Google Scholar). The GPM protein expectation score is a measure of the confidence of protein identification for which the lower the log value, the smaller the probability that this represents a random match. When identifying proteins in the MPR affinity-purified protein mixtures, the score of highest confidence obtained at random when searching a reversed orientation human proteome was used as the threshold for acceptance of protein assignments. Method-specific thresholds were determined for unfractionated digests (log(e) = −5.1 and −5.4 for the LTQ and Q-TOF, respectively) and for proteins fractionated by 1D PAGE when each gel slice was analyzed individually (log(e) = −4.3 and −4.1 for the LTQ and Q-TOF, respectively). Each threshold was used to filter the respective datasets obtained from searching the forward orientation human proteome. Samples were analyzed using different instruments (LTQ and Q-TOF), were singly and doubly purified, and were prepared for analysis using several different methods (solution digest and gel electrophoresis). Thus, as an additional level of stringency, proteins identified with one approach alone were rejected if the protein assignment scores failed to reach a threshold of log(e) less than −10. For analysis of purified Man-6-P glycopeptides, the threshold for acceptance of peptide assignments to proteins not previously classified as lysosomal was the score of lowest confidence of a deglycosylated peptide assigned to a known lysosomal species (peptide log(e) of −2.3 for β-glucuronidase) with the added stringency that peptides with log(e) more than −3 were only included if the assignment could be clearly verified following manual inspection. A summary of data obtained with each instrument with different samples is presented in Supplemental Table 1. For each protein identified, the sequence coverage and number of unique peptides assigned are summarized in Supplemental Table 2. xml files for each database search that include protein assignments and MS/MS spectra are also included as supplemental data. (Saved xml data can be viewed on line by uploading to the GPM (human.thegpm.org/tandem/thegpm_upview.html). Experimental parameters for each xml file are summarized in Supplemental Table 3. Man-6-P glycoproteins were initially purified from human plasma with a yield of ∼500 μg/liter, which represents ∼0.001% of the total starting protein content. Staining of the mixture after fractionation by two-dimensional gel electrophoresis using pH 3–10 isoelectric gradients revealed a complex pattern of purified proteins with >1500 spots resolved (Fig. 1A). A significant problem encountered using an affinity purification approach to identify lysosomal proteins based on the presence of Man-6-P is the identification of false positives (24Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. Carnemolla B. Orecchia P. Zardi L. Righetti P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1594) Google Scholar). Assuming correct protein assignment, false positives can represent abundant or “sticky” nonspecific contaminants (e.g. immunoglobulins or albumin) that bind nonspecifically and continuously leach off the column throughout the washing process and during the Man-6-P elution step. Alternatively non-Man-6-P proteins such as lectins or protease inhibitors can bind to and copurify with true Man-6-P glycoproteins, and these can be regarded as “specific contaminants” because they will elute specifically from the immobilized MPR with Man-6-P. A particular concern in this study, given that we expected lysosomal proteins to be very low abundance constituents of plasma, was the presence of nonspecific contaminants. To assess purity, we fractionated the mixture by two-dimensional gel electrophoresis, transferred the sample to nitrocellulose, and used a radiolabeled form of the sCI-MPR in a Western blot style assay (22Czupalla C. Mansukoski H. Riedl T. Thiel D. Krause E. Hoflack B. Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism.Mol. Cell. Proteomics. 2006; 5: 134-143Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) to visualize Man-6-P glycoproteins (Fig. 1B). When the resulting MPR blot was compared with the stained gel, it was clear that a considerable proportion of the purified proteins appeared not to contain Man-6-P. We therefore repeated the purification on our preparation and now found that most of spots corresponding to the twice-purified proteins appeared to contain Man-6-P (Fig. 1, C and D). Given the stringency of this double purification, many of the spots that appeared not to contain Man-6-P probably represent either specific contaminants or proteolytically processed chains of lysosomal proteins that do not contain Man-6-P rather than nonspecific contaminants. Protein yields after two rounds of purification were approximately half of those obtained after a single round of purification (on average, 230 μg/liter compared with 480 μg/liter), reflecting the loss of contaminants. A number of mass spectrometric" @default.
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• W2103064512 date "2006-10-01" @default.
• W2103064512 modified "2023-10-17" @default.
• W2103064512 title "Identification and Validation of Mannose 6-Phosphate Glycoproteins in Human Plasma Reveal a Wide Range of Lysosomal and Non-lysosomal Proteins" @default.
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