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• W2891568018 abstract "Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized for the enrichment of phosphopeptides using Fe3+-IMAC chromatography, that proteome-wide M6P glycopeptides can be selectively enriched and subsequently analyzed by mass spectrometry, taking advantage of exclusive phosphomannose oxonium fragment marker ions. As proof-of-concept of this protocol, applying it to HeLa cells, we identified hundreds of M6P-modified glycopeptides on 35 M6P-modified glycoproteins. We next targeted CHO cells, either wild-type or cells deficient in Acp2 and Acp5, which are acid phosphatases targeting M6P. In the KO CHO cells we observed a 20-fold increase of the abundance of the M6P-modification on endogenous CHO glycoproteins but also on the recombinantly over-expressed lysosomal human alpha-galactosidase. We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases. Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized for the enrichment of phosphopeptides using Fe3+-IMAC chromatography, that proteome-wide M6P glycopeptides can be selectively enriched and subsequently analyzed by mass spectrometry, taking advantage of exclusive phosphomannose oxonium fragment marker ions. As proof-of-concept of this protocol, applying it to HeLa cells, we identified hundreds of M6P-modified glycopeptides on 35 M6P-modified glycoproteins. We next targeted CHO cells, either wild-type or cells deficient in Acp2 and Acp5, which are acid phosphatases targeting M6P. In the KO CHO cells we observed a 20-fold increase of the abundance of the M6P-modification on endogenous CHO glycoproteins but also on the recombinantly over-expressed lysosomal human alpha-galactosidase. We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases. Lysosomes and lysosome associated organelles are cytosolic acidic compartments responsible for the degradation and digestion of a variety of biological macromolecules (1Saftig P. Klumperman J. Lysosome biogenesis and lysosomal membrane proteins: Trafficking meets function.Nat. Rev. Mol. Cell Biol. 2009; 10: 623-635Crossref PubMed Scopus (1106) Google Scholar). Its degradative activity originates from the presence of over 60 lysosomal acid hydrolases. Most of the lysosomal enzymes are incorporated into the organelle triggered by a specific post-translational modification involving a unique mannose-6-phosphate (M6P) 11 The abbreviations used are:M6PMannose-6-PhosphatePSMpeptide to spectrum matchManMannoseIMACimmobilized metal affinity chromatographyERendoplasmic reticulumGlcNAcN-acetylglucosamineUCEuncovering enzymeAcp2acid phosphatase 2Acp5acid phosphatase 5MSmass spectrometryHCDhigher energy c-trap dissociationEThcDelectron-transfer higher-energy collision dissociationKOknock out. moiety. This sugar moiety can be recognized by two distinct mannose-6-phosphate receptors in the Golgi complex, that subsequently transport their cargo toward the lysosome (2Dahms N.M. Lobel P. Kornfeld S. Mannose 6-phosphate receptors and lysosomal enzyme targeting.J. Biol. Chem. 1989; 264: 12115-12118Abstract Full Text PDF PubMed Google Scholar, 3Ghosh P. Dahms N.M. Kornfeld S. Mannose 6-phosphate receptors: new twist in the tale.Nat. Rev. Mol. Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (799) Google Scholar). Mannose-6-Phosphate peptide to spectrum match Mannose immobilized metal affinity chromatography endoplasmic reticulum N-acetylglucosamine uncovering enzyme acid phosphatase 2 acid phosphatase 5 mass spectrometry higher energy c-trap dissociation electron-transfer higher-energy collision dissociation knock out. Biosynthesis of the M6P tag starts in the rough endoplasmic reticulum (ER), where co-translational modification results in the N-glycosidic linkage of a 14-sugar glycan entity on selected asparagine residues in targeted proteins (Fig. 1.1 to 1.4). Subsequent removal of three glucose molecules by glucosidases and a terminal mannose moiety by mannosidase, produces uniform glycoproteins prepared for export from the ER, to the Golgi apparatus (Fig. 1.5 to 1.8) (4Lodish H. Berk A. Zipursky S.L. Matsudaira P. Baltimore D. Darnell J. Molecular Cell Biol. W. H. Freeman, New York2000Google Scholar). In the cis-Golgi network, selective phosphorylation of the proteins is achieved by the recognition of a lysine surface patch by the GlcNAc-phosphotransferase (5Dahms N.M. Olson L.J. Kim J.-J.P. Strategies for carbohydrate recognition by the mannose 6-phosphate receptors.Glycobiology. 2008; 18: 664-678Crossref PubMed Scopus (82) Google Scholar). The enzyme initiates a two-step reaction by catalyzing the addition of a GlcNAc-1-phosphate molecule to the outermost mannose residues (Fig. 1.9). The subsequent removal of the terminal GlcNAc group occurs by the GlcNAc-1-phosphodiester-N-acetylglucosaminidase (“uncovering enzyme” (UCE)), that finally produces the M6P that is recognized by the mannose-6-phosphate receptors. In the trans-Golgi network, the mannose-6-phosphate receptors recognize and bind to the M6P sugar moiety initiating the incorporation of newly synthesized lysosomal acid hydrolases in clathrin coated vesicles for their ultimate delivery to the lysosomes. Here, the mannose-6-phosphate receptor substrates dissociate from their receptor because of the substantial drop in pH, upon which many proteins undergo additional modifications to produce a fully functional enzyme. Some of such modifications are activated by the phosphatases Acp2 and Acp5 that remove phosphate groups from lysosomal acid hydrolases, triggering them to execute their function as hydrolases, activating the degradation of macromolecules (6Makrypidi G. Damme M. Müller-Loennies S. Trusch M. Schmidt B. Schlüter H. Heeren J. Lübke T. Saftig P. Braulke T. Mannose 6 dephosphorylation of lysosomal proteins mediated by acid phosphatases Acp2 and Acp5.Mol. Cell. Biol. 2012; 32: 774-782Crossref PubMed Scopus (35) Google Scholar). Providing lysosomes with the correct array of enzymes is of uttermost importance in cellular function. Absence or improper activity of one of the lysosomal acid hydrolases results in diverging phenotypes ranging from improper antigen processing because of improper cathepsin function (7Blum J.S. Wearsch P.A. Cresswell P. Pathways of Antigen Processing.Annu. Rev. Immunol. 2013; 31: 443-473Crossref PubMed Scopus (929) Google Scholar, 8Turk V. Turk B. Gunčar G. Turk D. Kos J. Lysosomal cathepsins: Structure, role in antigen processing and presentation, and cancer.Adv. Enzyme Regul. 2002; 42: 285-303Crossref PubMed Scopus (158) Google Scholar, 9Manoury B. Proteases: Essential actors in processing antigens and intracellular toll-like receptors.Front. Immunol. 2013; 4: 1-5Crossref PubMed Scopus (23) Google Scholar) all the way to lysosomal storage diseases. The most prominent phenotype of lysosomal storage diseases is the accumulation of glycoproteins, unprocessed lipids, mucopolysaccharides, and combinations thereof. Two well-known examples include Fabry's and Gaucher disease, resulting from the absence of alpha-galactosidase A and glucocerebrosidase enzymes, respectively, causing sphingolipid accumulation inside the lysosome (10Boustany R.M.N. Lysosomal storage diseases – The horizon expands.Nat. Rev. Neurol. 2013; 9: 583-598Crossref PubMed Scopus (168) Google Scholar, 11Platt F.M. Sphingolipid lysosomal storage disorders.Nature. 2014; 510: 68-75Crossref PubMed Scopus (221) Google Scholar). Currently, the only therapeutic option for such disorders is enzyme replacement therapy where a recombinant version of the enzyme is provided. One of the critical attributes of such replacement therapies in the presence of the M6P-moitey on the enzyme, ensuring proper targeting to the lysosome where they can exert their therapeutic function. To understand the origin of lysosomal storage diseases, it is essential to obtain an overview of the wide range of proteins that are contained within the lysosome. The current most widely used technique in the proteomic analysis of the lysosome is the affinity purification from tissue samples using columns with immobilized receptors (12Qian M. Sleat D.E. Zheng H. Moore D. Lobel P. Proteomics analysis of serum from mutant mice reveals lysosomal proteins selectively transported by each of the two mannose 6-phosphate receptors.Mol. Cell. Proteomics. 2008; 7: 58-70Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 13Lübke T. Lobel P. Sleat D.E. Proteomics of the lysosome.Biochim. Biophys. Acta - Mol. Cell Res. 2009; 1793: 625-635Crossref PubMed Scopus (191) Google Scholar, 14Sleat D.E. Valle M.C.D. Zheng H. Moore D.F. Lobel P. The mannose 6-phosphate glycoprotein proteome.J. Proteome Res. 2008; 7: 3010-3021Crossref PubMed Scopus (53) Google Scholar, 15Sleat D.E. Sun P. Wiseman J.A. Huang L. El-Banna M. Zheng H. Moore D.F. Lobel P. Extending the mannose 6-phosphate glycoproteome by high resolution/accuracy mass spectrometry analysis of control and acid phosphatase 5-deficient mice.Mol. Cell. Proteomics. 2013; 12: 1806-1817Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In these earlier used methods, M6P-modified proteins were extracted, deglycosylated with peptide N-glycosidase F and subsequently subjected to mass spectrometry (MS) analysis. However, these approaches can result in false positive identifications because of the applied removal of the M6P-tag. A few studies focused on intact M6P glycopeptides, although they were mainly focused on the analysis of a single lysosomal protein (16Park H. Kim J. Lee Y.K. Kim W. You S.K. Do J. Jang Y. Oh D.B. Il Kim J. Kim H.H. Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease.Biochem. Biophys. Res. Commun. 2018; 495: 2418-2424Crossref PubMed Scopus (12) Google Scholar, 17McVie-Wylie A.J. Lee K.L. Qiu H. Jin X. Do H. Gotschall R. Thurberg B.L. Rogers C. Raben N. O'Callaghan M. Canfield W. Andrews L. McPherson J.M. Mattaliano R.J. Biochemical and pharmacological characterization of different recombinant acid α-glucosidase preparations evaluated for the treatment of Pompe disease.Mol. Genet. Metab. 2008; 94: 448-455Crossref PubMed Scopus (74) Google Scholar). Here we use a well-known enrichment method, for phosphopeptides, based on iron immobilized metal ion affinity chromatography (Fe3+-IMAC) that we show enables the co-enrichment of M6P-modified glycopeptides from a complex cell lysate. To distinguish the M6P-modified glycopeptides from phosphopeptides we utilize a specific signature fragment ion of M6P (m/z 243.026) (18Zhang Y. Go E.P. Jiang H. Desaire H. A novel mass spectrometric method to distinguish isobaric monosaccharides that are phosphorylated or sulfated using ion-pairing reagents.J. Am. Soc. Mass Spectrom. 2005; 16: 1827-1839Crossref PubMed Scopus (15) Google Scholar) observed during higher-energy c-trap dissociation (HCD). Observation of this ion in HCD triggers electron-transfer/higher-energy collision dissociation (EThcD), which results in a more confident characterization of the exact sites of the M6P-modified glycopeptides, and provides signature glycan fragments enabling confident glycoform identification. Initial analysis of a HeLa cell lysate with our modified enrichment and targeted MS approach resulted in the identification of 35 M6P-modified glycoproteins and 46 M6P glycosites from hundreds of detected M6P glycopeptides. Next, we applied our approach to wild-type CHO cells and CHO cells carrying a gene knockout (KO) of the acid phosphatases Acp2 and Acp5, which dephosphorylate lysosomal proteins. KO of these phosphatases resulted in >20 fold increase in abundance of M6P glycopeptides, enabling a deeper coverage of the CHO M6P-glycoproteome. Finally, we demonstrate that recombinant expression of lysosomal human alpha-galactosidase in WT CHO cells and CHO cells carrying the Acp2 and Acp5 KO results in a drastic increase of the M6P-moiety on alpha-galactosidase in the KO CHO cell line. We conclude that Fe3+-IMAC enables facile enrichment of intact M6P glycopeptides and could be used to extend biological insight gained from standard phosphoproteomic studies, additionally, when coupled with glycoengineering strategies it can be used for characterization of improved therapeutic enzymes used for the treatment of lysosomal storage diseases. HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen, Landsmeer, the Netherlands) and 0.05 mg/ml penicillin/streptomycin (Invitrogen) at 37 °C in 5% CO2 in 15 cm plate. Cells were collected at 80% confluence by centrifugation and washed three times with ice-cold PBS. A CRISPR/Cas9 based approach was used for the gene knockout (KO) in CHO cells as described preciously (19Schulz M.A. Tian W. Mao Y. Van Coillie J. Sun L. Larsen J.S. Chen Y.-H. Kristensen C. Vakhrushev S.Y. Clausen H. Yang Z. Glycoengineering design options for IgG1 in CHO cells using precise gene editing.Glycobiology. 2018; 28: 1-8Crossref Scopus (22) Google Scholar). CHOZN GS−/− cells with stable expression of alpha-galactosidase were used as the parental clone (as below: wild type) for the Acp2/5 KO. Cells were maintained as suspension cultures in EX-CELL CHO CD Fusion serum-free media (Sigma-Aldrich, Brøndbyvester, Denmark) in 50 ml TPP TubeSpin® Bioreactors with 180 rpm shaking speed at 37 °C and 5% CO2. Cells were seeded at 0.5 × 106 cells/ml in T25 flask (NUNC, Hvidovre, Denmark) 1 day prior to transfection. Electroporation was conducted with 2 × 106 cells with a DNA mixture of 1 μg of Cas9-GFP plasmid and 1 μg of gRNA plasmid (U6GRNA, Addgene Plasmid #68370) using an Amaxa kit V and program U24 with Amaxa Nucleofector 2B (Lonza, Copenhagen, Denmark). Forty-eight hours after nucleofection the 10–15% highest GFP expression pools of cells were enriched by FACS, and after 1 week cultured cells were single-cell sorted by FACS into 96-wells. KO clones were identified by Indel Detection by Amplicon Analysis (IDAA) as described (20Yang Z. Steentoft C. Hauge C. Hansen L. Thomsen A.L. Niola F. Vester-Christensen M.B. Frödin M. Clausen H. Wandall H.H. Bennett E.P. Fast and sensitive detection of indels induced by precise gene targeting.Nucleic Acids Res. 2015; 43: e59Crossref PubMed Scopus (115) Google Scholar). Selected clones were further verified by Sanger sequencing. Around 1.5 × 108 of wild type and KO cells were collected by centrifugation and washed three times with ice-cold PBS. For both Hela and CHO cells, three technical replicates were performed. The values of peptide to spectrum matches (PSM) from each detected glycan composition across all M6P-modified peptides were summed then mean ± standard deviation of three technical replicates were calculated. HeLa, CHO wild type and CHO KO cell pellets were re-suspended in lysis buffer containing 100 mm Tris-HCl (pH 8.5) (Sigma-Aldrich), 7 m urea (Sigma-Aldrich), 5 mm Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Sigma-Aldrich), 30 mm chloroacetamide (CAA, Sigma-Aldrich), Triton X-100 (1%) (Sigma-Aldrich), 2 mm magnesium sulfate (Sigma-Aldrich), Benzonase (1%) (Merck Millipore, Darmstadt, Germany), phosphoSTOP (Roche) and complete mini EDTA free (Roche, Woerden, the Netherlands). Then, cells were disrupted by sonication for 45 min (20 s on, 40 s off) using a Bioruptor Plus (Diagenode, Seraing, Belgium). Cell debris was removed by centrifugation at 14,000 rpm for 1 h at 4 °C and the supernatant was kept. Impurities were removed by methanol/chloroform protein precipitation as follows: 1 ml of supernatant was mixed with 4 ml of methanol (Sigma-Aldrich), 1 ml chloroform (Sigma-Aldrich) and 3 ml ultrapure water with thorough vortexing after each addition. The mixture was then centrifuged for 10 min at 5000 rpm at room temperature (RT). The upper layer was discarded, and 3 ml of methanol was added. After sonication and centrifugation (5000 rpm, 10 min at RT), the solvent was removed, and the precipitate was allowed to air dry. The pellet was resuspended in digestion buffer containing 100 mm Tris-HCl (pH 8.5), 0.5% Rapigest (Water, Etten-Leur, the Netherlands), 5 mm TCEP and 30 mm CAA. Trypsin (Sigma-Aldrich) and Lys-C (Wako, Neuss, Germany) were added to a 1:50 and 1:100 ratio (w/w), respectively. Digestion was performed overnight at 37 °C. After acidic precipitation of Rapigest with 0.5% trifluoroacetic acid (TFA), peptides were cleaned by Sep-Pak C18 1cc vac cartridge, dried and stored at −80 °C until Fe3+-IMAC enrichment. Fe3+-IMAC was performed in technical triplicates for each biological group as described previously (21Potel C.M. Lin M.-H. Heck A.J.R. Lemeer S. Defeating major contaminants in Fe 3+-IMAC phosphopeptide enrichment.Mol. Cell. Proteomics. 2018; (mcp.TIR117.000518)Abstract Full Text Full Text PDF Scopus (50) Google Scholar). Tryptic peptides (2 mg) were re-suspended with ice-cold buffer A and the pH was adjusted to 2.3 using 10% TFA before injection into the Fe3+-IMAC column (Propac IMAC-10 4 × 50 mm column, ThermoFisher Scientific, Landsmeer, the Netherlands). Mobile-phase solvent A consisted of 30% acetonitrile and 0.07% TFA, and mobile-phase solvent B consisted of 0.3% NH4OH in water. Loading was performed at a flow rate of 0.1 ml/min for 7 min with 0% B and nonphosphorylated or nonphosphomannosylated peptides were washed out using a flow rate of 1 ml/min for 5 min with 0% B. Phosphopeptides and phosphomannose peptides were eluted at a flow rate of 1 ml/min with 50% B for 1.5 min, followed 0.5 ml/min for 2.5 min with 50% B, and finally held at 1 ml/min of 0% B for 9 min. Collected phosphopeptides and phosphomannose peptides were dried down using a lyophilizer. Nanoflow LC-MS/MS was performed by coupling an Agilent 1290 (Agilent Technologies, Middelburg, Netherlands) to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific, Bremen, Germany) for the analysis of peptides enriched by Fe3+-IMAC. After resuspension in 0.1% TFA, peptides were separated by using a 100 μm inner diameter 2 cm trap column (in-house packed with ReproSil-Pur C18-AQ, 3 μm) (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) coupled to a 50 μm inner diameter 50 cm analytical column (in-house packed with Poroshell 120 EC-C18, 2.7 μm) (Agilent Technologies, Amstelveen, The Netherlands). Mobile-phase solvent A consisted of 0.1% FA in water, and mobile-phase solvent B consisted of 0.1% FA in ACN. Trapping was performed at a flow rate of 5 μl/min for 5 min with 0% B and peptides were eluted using a passively split flow of 300 nl/min for 85 min with 8% to 40% B over 75 min, 40% to 100% B over 3 min, 100% B for 1 min, 100% to 0% B over 1 min, and finally held at 0% B for 10 min. Peptides were ionized using 2.0 kV spray voltage and a capillary temperature of 320 °C. The mass spectrometer was set to acquire full-scan MS spectra (375–1700 m/z) for a maximum injection time of 50 ms at a mass resolution of 60,000 and an automated gain control (AGC) target value of 4e5. The dynamic exclusion was set to 20s at exclusion window of 10 ppm with a cycle time of 3s. Charge-state screening was enabled, and precursors with +2 to +6 charge states and intensities >1e5 were selected for tandem mass spectrometry (MS/MS). HCD MS/MS (150–1800 m/z) acquisition was performed in the HCD cell, with the readout in the Orbitrap mass analyzer at a resolution of 30,000 (isolation window of 1.6 Th) and an AGC target value of 5e4 or a maximum injection time of 75 ms with a normalized collision energy of 30%. When the oxonium ion of phosphomannose (Man P, 243.026+) was observed, EThcD MS/MS on the same precursor was triggered (isolation window of 1.6 Th) and fragment ions (150–2100 m/z) were analyzed in the Orbitrap mass analyzer at a resolution of 30,000, AGC target value of 2e5 or a maximum injection time of 200 ms with activation of ETD and supplemental activation with a normalized collision energy of 27%. Raw data files of peptides enriched by Fe3+-IMAC were processed using Byonic software (ver 2.15.10) (Protein Metrics Inc.) with the following search parameters: trypsin digestion with a maximum of 2 missed cleavages, unreviewed CHO database (Uniprot, 34 962 entries,March 2018), and reviewed human database for HeLa samples (Uniprot, 26 339, March 2018). Precursor ion mass tolerance, 10 ppm; fragmentation type, both HCD & EThcD; product ion mass tolerance for HCD, 20 ppm; product mass tolerance for EThcD, 20 ppm; carbamidomethylation of cysteines as a fixed modification; variable modifications: methionine oxidation, phosphorylation on serine, threonine and tyrosine residues. Byonic database of the 50 common biantennary N-glycans and 20 M6P-modified N-glycans (full list of M6P compositions is provided in supplemental Table S1). Byonic cut-off score of 100 was used and all M6P-modified identified glycopeptide spectra were further manually validated for the presence of the signature ion (243.026 m/z). Skyline (Skyline-daily, Version 4.0.9.11664) was used to build a spectral library from search results and extracted ion chromatograms (XICs) of precursor ions with the first five isotopes being used to calculate peak areas of selected peptides shown in the Fig. 5, Fig. 6. Raw files of previously published study (22Post H. Penning R. Fitzpatrick M.A. Garrigues L.B. Wu W. Macgillavry H.D. Hoogenraad C.C. Heck A.J.R. Altelaar A.F.M. Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.J. Proteome Res. 2017; 16: 728-737Crossref PubMed Scopus (71) Google Scholar) of three standard approaches for phosphopeptide enrichment were downloaded from PRIDE partner (identifier: PXD005366) and searched with same above-mentioned parameters in Byonic software except fragmentation type was only HCD.Fig. 6Qualitative and quantitative overview of the M6P-modifications found on Human alpha-galactosidase (GLA) when expressed in CHO WT or CHO Acp2/5 KO cells. A, Comparison of glycan compositional profiles observed in CHO WT (gray) and CHO Acp2/5 KO (black) samples. Error bars denote standard deviation between replicates. B, Comparison of abundances of an unmodified tryptic peptide (left) and M6P-modified glycopeptide (right) of GLA expressed in CHO WT and CHO Acp2/5 KO cells, reveals similar expression, but distinct higher (∼20 fold) M6P-modification.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Somewhat inspired by studies (24Larsen M.R. Jensen S.S. Jakobsen L.A. Heegaard N.H.H. Exploring the Sialiome Using Titanium Dioxide Chromatography and Mass Spectrometry.Mol. Cell. Proteomics. 2007; 6: 1778-1787Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 25Palmisano G. Lendal S.E. Engholm-Keller K. Leth-Larsen R. Parker B.L. Larsen M.R. Selective enrichment of sialic acid-containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry.Nat. Protoc. 2010; 5: 1974-1982Crossref PubMed Scopus (203) Google Scholar, 26Melo-Braga M.N. Schulz M. Liu Q. Swistowski A. Palmisano G. Engholm-Keller K. Jakobsen L. Zeng X. Larsen M.R. Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells.Mol. Cell. Proteomics. 2014; 13: 311-328Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) demonstrating the applicability of common phosphopeptide enrichment protocols to enrich also sialylated glycopeptides we investigated whether also M6P-modified glycopeptides could be enriched. To this end we first set out to reanalyze previously published data (22Post H. Penning R. Fitzpatrick M.A. Garrigues L.B. Wu W. Macgillavry H.D. Hoogenraad C.C. Heck A.J.R. Altelaar A.F.M. Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.J. Proteome Res. 2017; 16: 728-737Crossref PubMed Scopus (71) Google Scholar) in which three standard approaches for phosphopeptide enrichment were compared: Ti4+ and Fe3+ based IMAC, and TiO2 based enrichment, now allowing variable M6P-modifications, GlcNAc2Man3–8Phospho1–2 and GlcNAc3–4Man6–8Phospho1NeuAc0–1 (supplemental Table S1), on Asn residues in addition to phosphorylation on Ser, Thr and Tyr. Our reanalysis of these data sets (Fig. 2) revealed that next to several thousands of phosphopeptides also tens of M6P-modified glycopeptides were co-enriched. As previously noted (22Post H. Penning R. Fitzpatrick M.A. Garrigues L.B. Wu W. Macgillavry H.D. Hoogenraad C.C. Heck A.J.R. Altelaar A.F.M. Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.J. Proteome Res. 2017; 16: 728-737Crossref PubMed Scopus (71) Google Scholar) the three enrichment approaches performed equally well for the phosphopeptides with a great overlap in identified unique phosphopeptides. In the co-enrichment of M6P-modified glycopeptides, we seemingly did observe a bias, whereby the Fe3+-IMAC based enrichment provided the highest number of identifications with 25 identified M6P-modfied glycopeptides. Ti4+ IMAC provided a slightly lower efficiency with 14 identified M6P glycopeptides, and more surprisingly, only 3 M6P glycopeptides were identified by using the TiO2 based enrichment. Although in this experiment Fe3+-IMAC and Ti4+ IMAC show slightly different performance, we identified the same glycoforms, GlcNAc2Man6–7Phospho1–2, albeit on a different peptide backbones indicating that the difference is likely because of stochastic nature of precursor ion selection during MS analysis. From these experiments, we decided to perform the rest of the experiments using Fe3+-IMAC and we sought to further optimize the enrichment and analysis protocol to extend the coverage of M6P-modified glycoproteome. We hypothesized that there are two main reasons for the low numbers of M6P-modified glycopeptides. First, the MS fragmentation scheme based only on HCD is not optimal for glycopeptide identifications because of the preferential cleavage of the glycan moiety (27Thaysen-Andersen M. Packer N.H. Schulz B.L. Maturing glycoproteomics technologies provide unique structural insights into the N -glycoproteome and its regulation in health and disease.Mol. Cell. Proteomics. 2016; 2 (mcp.O115.057638)Google Scholar, 28Hu H. Khatri K. Klein J. Leymarie N. Zaia J. A review of methods for interpretation of glycopeptide tandem mass spectral data.Glycoconj. J. 2015; 33: 1-12PubMed Google Scholar). Second, combination of low starting material used in (22Post H. Penning R. Fitzpatrick M.A. Garrigues L.B. Wu W. Macgillavry H.D. Hoogenraad C.C. Heck A.J.R. Altelaar A.F.M. Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.J. Proteome Res. 2017; 16: 728-737Crossref PubMed Scopus (71) Google Scholar) and the inherent low abundance of M6P glycoproteins probably precluded their detection during MS analysis. To test our hypothesis, we performed an experiment where we increased the amount of starting material to 2 mg of protein and optimized the MS fragmentation scheme to combine HCD with EThcD fragmentation, which were triggered upon observation of the signature fragment ion in the HCD spectra for M6P-modified glycopeptides, namely the phosphomannose oxonium ion (243.026 m/z) (18Zhang Y. Go E.P. Jiang H. Desaire H. A novel mass spectrometric method to distinguish isobaric monosaccharides that are phosphorylated or sulfated using ion-pairing reagents.J. Am. Soc. Mass Spectrom. 2005; 16: 1827-1839Crossref PubMed Scopus (15) Google Scholar). Using this approach, we could now identify almost a hundred of unique M6P-modified glycopeptides (supplemental Table S2) in Hela cells corresponding to 35 M6P-modified glycoprotein and 46 M6P glycosites (Table I), which is significantly more than what we could identify from the published dataset (22Post H. Penning R. Fitzpatrick M.A. Garrigues L.B. Wu W. Macgillavry H.D. Hoogenraad C.C. Heck A.J.R. Altelaar A.F.M. Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.J. Proteome Res. 2017; 16: 728-737Crossref PubMed Scopus (71) Google Scholar) (Fig. 2C). We observed that the pool of M6P glycopeptides in frequency comprise around 2–3% of the total peptide (M6P glycopeptide and phosphopeptide) pool. Manual inspection of the" @default.
• W2891568018 created "2018-09-27" @default.
• W2891568018 creator A5001199496 @default.
• W2891568018 creator A5019159472 @default.
• W2891568018 creator A5036297122 @default.
• W2891568018 creator A5038148203 @default.
• W2891568018 creator A5053603496 @default.
• W2891568018 creator A5058714848 @default.
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• W2891568018 date "2019-01-01" @default.
• W2891568018 modified "2023-10-18" @default.
• W2891568018 title "Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification" @default.
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