PMCID: PMC4184449

 

    Legend: Gene, Sites, Suger

Section : Glycopeptide Identification Based on AccurateIntact Mass

Content :

1. We compared the usefulness of LC–MS dataacquired usingHILIC-C18-MS versus C18-MS for profiling glycoprotein proteolyticpeptides
2. For this purpose, the neutral mass values extracted fromthe data sets were searched against a set of theoretical glycopeptidemasses , with a 10 ppm mass-error tolerance
3. The theoretical massesconsisted of the protein proteolytic peptides with up to two missedcleavage sites
4. Masses for those peptides containing an N-glycosylation sequon were calculated as a set of glycosylation variantsusing N-glycosylation com positions ranging from core N-glycan structures to penta-antennary complex-type N-glycans containing N-acetylneuraminicacid and high-mannose N-glycans (shown in Supporting Information Section S-3)
5. The GlycReSoftprogram was used to match and score deconvolutedmasses from an LC–MS experiment against this list of theoreticalcompound accurate masses
6. Because the complexity in glycopeptide dataincreases with the number of glycosylation sites , intact mass assignmentssuffer from ambiguous matches and false positives that cannot be verifiedin the absence of tandem MS. We acquired LC–MS profilingdata for three glycoproteins with a range of complexities
7. Transferrinis known to have two N-linked glycosylation siteswith complex N-glycans, and the glycan heterogeneityfor this protein is fairly limited with [5,4,0,2,0] [Hex, HexNAc,dHex, NeuAc, NeuGc] contributing to over 90% of the glycan distribution
8. Thus, this glycoprotein presents low complexity,which makes the search space small and minimizes the chances for falsepositives and ambiguous assignments
9. AGP, by contrast, has 5 known N-linked glycosylation sites with multiple genetic variants and a more diverse set of complexglycans that make this a relatively more complex glycoprotein
10. Recombinanthemagglutinin from A/USSR/90/1977 (H1N1), referredto herein as HA, was the most complex of the three glycoproteins analyzed,with 9 consensus glycosylation sites
11. In addition, theoretical trypticcleavage of HA produced 2 peptides that presented more than 1 putativeglycosylation site on a single peptide
12. Thus, expected ambiguity andfalse positives scaled with increasing number of glycosylation sitesin transferrin , AGP, and HA
13. The number of theoretical glycopeptidecompositions positions used for each glycoprotein is given in Table 1
14. We observed a significantly higher number of glycopeptide matchesusing HILIC-C18 LC–MS data over C18 LC–MS data, forall glycoproteins tested
15. Table 1 shows thenumber of glycopeptides and peptides identified based on intact glycopeptidemasses
16. Even with the limited possibility of false matches when searchingsolely based on theoretical masses, it was evident that HILIC-C18enriched glycopeptides efficiently
17. Data were acquired as analyticaltriplicates, and peptide/glycopeptide com positions found in all threereplicates were considered matches

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Section : Oxonium Ion Distributions

Content :

1. In order to confirm the ioncom positions from the MS data, we used data-dependent LC–tandemmass spectrometry
2. Collisional dissociation of glycopeptides leadsto formation of oxonium ions that are useful as features for identifyingglycopeptides in LC–MS/MS chromatograms
3. We therefore used the absence of oxonium ions to disqualifya precursor ion as glycopeptide
4. On the basis of this rationale, wecompared the abundances of oxonium ions in extracted ion chromatograms(EICs) of glycopeptides observed using C18-MS/MS versus HILIC-C18-MS/MSdata
5. Figure 1 compares the HexNAc oxoniumion (m/z 204) EIC with the BPC (basepeak chromatogram) for hemagglutinin
6. Oxonium ion profiles for therest of the proteins analyzed are presented in Supporting Information Figure S-1
7. When using HILIC-C18, weobserved that the abundances of the HexNAc (m/z 204) oxonium ion profile were similar to those of theBPC, consistent with the fact that most of the detected ions correspondedto glycopeptides
8. For the C18 LC–MS experiments, the abundancesof HexNAc oxonium ion were 10-fold lower than the BPC, indicatingthat most of the detected ions corresponded to unglycosylated peptides
9. Using C18-MS, unmodified peptides were the most abundant ions selectedby the acquisition software for tandem MS based on abundance
10. By contrast,for HILIC-C18 data, glycopeptides were the most abundant ions presentand were selected automatically for tandem MS. The oxonium ion distributionswere consistent with the results obtained from the LC–MS data,shown in Table 1, indicating the contributionof glycopeptides to total ion abundances in C18 and HILIC-C18 runs
11. Oxonium ions detectedearly in the chromatogram using HILIC-C18-MSresulted from a small fraction of the enriched glycopeptides fromthe HILIC trapping column getting washed away with the dead volumecontaining high organic mobile phase
12. This was a fixed and unbiasedloss that accounted for less that 2% of the total sample abundanceand did not affect the relative abundances of the compounds beingretained on the C18 analytical columns
13. Figure 2 shows the percent of precursorions identified as glycopeptides based on formation of oxonium ionsin data-dependent LC–MS/MS runs using HILIC-C18-MS versus C18-MS
14. We concluded that use of HILIC-C18-MS resulted in significant increasein the ability to analyze glycopeptides using data-dependent LC–MS/MS
15. These data demonstrate that the HILIC-C18-MS increased abundancesof glycopeptides relative to unglycosylated peptides and thus thequality of data-dependent LC–tandem MS data
16. The improved dataquality increased confidence in assignments and decreased false identifications
17. Two AGP glycopeptides (ENGTISR/ENGTVSRand NEEYNK) were detectedonly using HILIC-C18-MS
18. This was due to the fact that tryptic cleavageof AGP produces glycopeptides that are only 5–7 amino acidslong and are not retained during the trapping step when using C18-MS
19. These glycopeptides were trapped on the HILIC enrichment column whenusing HILIC-C18-MS and eluted soon after the solvent front on theanalytical chromatogram, as seen in SupportingInformation Figure S-1(b), which allowed them to undergo MSand MS/MS analysis
20. The C18 trapping step can be eliminated to retainany shorter glycopeptides in a sample
21. However, in our experience,an online trapping step is a useful means to eliminate salts and othercontaminants while minimizing manual manipulation

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Section : Tandem MS ofGlycopeptides

Content :

1. While the presence of oxoniumions from dissociation of glycosidic bonds is a useful feature, theformation of peptide backbone product ions is necessary for unambiguousassignment of the peptide sequence and glycan com position
2. We thereforeinvestigated the extent to which the increased glycopeptide precursorion abundances obtained using HILIC-C18-MS improved the quality ofthe resulting glycopeptide tandem mass spectra
3. Collisional dissociationusing typical conditions for peptide tandem MS results in formationof abundant ions from dissociation of the glycan with those from peptidebackbone dissociation often not detected
4. In the interest of maximizingthe information produced from a CAD LC–MS/MS experiment, weinvestigated use of higher collision energies for glycopeptides
5. Figures 3 and S-2 show examplesof glycopeptide tandem mass spectra from each of the three glycoproteinsstudied; collision energies were calculated as per the equations describedin Supporting Information Section S-1
6. As expected, glycopeptide dissociation produced abundant oxonium ionsin the low m/z range that confirmedthe presence of monosaccharides in the precursor ion com position
7. In addition, peptide backbone product ions were detected that enableddirect identification of the peptide
8. This information increased confidencein true matches and decreased those in incorrect matches
9. This wasimportant in cases where more than one theoretical glycopeptide matchedan observed mass value
10. Complete peptide backbone or parts of thepeptide backbone could be sequenced for glycopeptide precursors , asshown in the annotated spectra
11. Intact peptide and glycopeptides +saccharide (referred to as stub glycopeptide ) ions were also detectedin the higher m/z range of the spectrathat matched the masses of peptide with N-glycancore structures
12. The presence of intact peptide or stub glycopeptideions significantly increased confidence of assignments over thosemade from MS-only data
13. In the majority of LC–tandem mass spectra,ions corresponding to peptide backbone product ions plus a HexNAcresidue were detected, which confirmed the site of glycosylation,as shown in Supporting Information Figure S-2 and Tables 2 and S-2
14. Although the relative abundanceof a glycopeptide precursor ionhad an effect on the quality of tandem MS by affecting the abilityto select the ion in a data-dependent LC–MS/MS experiment,the absolute abundance did not appear to play a role in generationof useful fragment ions for confident assignment of the glycopeptides
15. In order to demonstrate that the detection of peptide backbone productions was a universal phenomenon, we analyzed glycopeptides with differentpeptide backbones and varying absolute abundances from each of thethree glycoproteins studied
16. Table 2 summarizesthe types of product ions observed using HILC-C18 vs C18 LC–tandemMS
17. Mass tables with observed and calculated fragment ion masses areshown in Supporting Information Section S-4
18. For all glycopeptide assignments that were confirmed by tandemMS, the following criteria were used: (1) intact glycopeptide massmatch, (2) presence of oxonium ions, (3) presence of either an abundantprotonated peptide ion and/or a peptide + HexNAc ion, and (4) presenceof significant peptide backbone product ion coverage
19. It is significantthat some of the glycopeptide com positions that were assigned to high-abundance precursor ions based on intact mass that also produced oxonium ionswere rejected (shown in red font) because the tandem MS stub glycopeptideor peptide backbone ions were not consistent with the assignment
20. This emphasizes the need for tandem MS on glycopeptides for confidentassignments
21. Although the ions shown in Table 2 were quite abundant in the mixtures analyzed, many were not selectedfor data-dependent tandem MS when using C18-MS due to the presenceof more abundant nonglycosylated peptides
22. In addition, GlycReSoftfailed to find and match some of these com positions in C18 data, whichwas probably due to very low abundances or presence of overlappingisotopic peaks
23. HILIC-C18-MS improved the abundances of glycopeptidesrelative to nonglycosylated peptides , which allowed these ions tobe matched by the GlycReSoft software
24. Data-dependent acquisitionof tandem mass spectra led to systematic identification of these glycopeptidesand allowed confident assignments due to the presence of peptide backboneproduct ions
25. The abundant product ions containing stub glycopeptidesand oxonium ions were useful for confirming peptide identities

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Section : Results for Transferrin

Content :

1. Transferrin presents two glycosylationsites, with the most abundant N-linked glycan ofcom position [5,4,0,2,0] at each of those two sequons
2. In agreementwith this, glycopeptides QQQHLFGSNVTDCSGNFCLFR-[5,4,0,2,0]and CGLVPVLAENYNK-[5,4,0,2,0] could be identified using tandemMS in both HILIC-C18 and C18 analyses
3. Other transferrin glycopeptides ,although identified by GlycReSoft in the C18 LC–MS data, didnot get selected for data-dependent tandem MS due to presence of abundantnonglycosylated peptides
4. However, these glycopeptides underwent tandemMS and could be confidently assigned in case of HILIC-C18

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Section : Resultsfor Alpha-1-acid Glycoprotein

Content :

1. Compared to transferrin ,which has only two glycosylation sites and limited glycan heterogeneity,AGP is more complex with five different glycosylation sites and differencesin peptide backbone arising from genetic variants
2. The HILIC-C18 methodwas significantly more effective at acquiring data-dependent tandemmass spectra of sufficient quality to enable identification of thepeptide backbone
3. We observed formation of useful peptide backboneions, even for precursor ions with moderate abundances (∼1–2× 105 counts)
4. Tandem mass spectra were useful indiscriminating single amino acid genetic variants ; for example, inTable 2, two AGP glycopeptides have similarpeptide sequences , ENGTVSR and ENGTISR, for which we observed completepeptide sequences in the glycopeptide tandem mass spectra of precursors1070.6586 (4+) and 1074.1627 (4+)
5. These peptides were not retainedusing the C18-MS system

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Section : Results for Influenza A Virus Hemagglutinin

Content :

1. HA presentsa considerably greater analytical challenge than AGP
2. Due to the presenceof 9 putative glycosylation sites with a wide distribution of possibleglycan com positions at each site, the number of theoretical glycopeptides (the search space) was more than 10-fold greater than that of AGP,as shown in Table 1
3. In addition, the numberof glycopeptide glycoforms detected was greater, and as a result ofhigher glycoform heterogeneity, the corresponding relative abundancesof each glycopeptide precursor ion were generally lower than thoseobserved for transferrin and AGP
4. In Table 2, we compared 5 different glycopeptide com positions that were identifiedby GlycReSoft in both HILIC-C18 and C18 data from HA analyses
5. Outof the 5 glycopeptides assigned by intact mass, only 1, NGSYPNLSK-[5,4,1,1,0],underwent tandem MS with C18 analysis
6. However, in the case of HILIC-C18,useful tandem MS data were acquired on all 5 glycopeptides , leadingto formation of not only peptide backbone ions but also backbone ionswith an attached HexNAc , facilitating identification of the glycosylationsite
7. This was important in case of NGSYPNLSK-[5,4,1,1,0], where twoglycosylation sequons, NGS and NLS, were present on the same glycopeptide ;HILIC-C18 tandem MS data allowed us to identify NLS as the occupiedsequon, as shown in Supporting Information TableS-3(c) and Figure 3
8. Tandem MS was alsouseful in identifying other modifications
9. For example, the exactsite of deamidation could be identified in case of HA glycopeptideNVTVTHSVNLLEDSHGK(1-Deamidation)-[7,6,1,3,0]
10. As shown in Table 1, measurement of glycopeptidemass did not suffice to identify the peptide and glycan com position because of the size of the search space
11. Thus, observed masses consistentwith more than one glycopeptide were not uncommon
12. We showed thattandem MS could be used to resolve ambiguities
13. As shown in Table 2, HA precursor ion m/z 1339.1270 (5+) matched three different com positions, SWSYIAETPNSENGTCYPGYFADYEELR-[7,6,1,3,0],NGSYPNLSKSYVNNKEK (1 deamidation)-[14,8,0,3,0], and NGSYPNLSKSYVNNK(2 deamidation)-[12,13,3,0,0], within a 10 ppm mass error tolerance
14. Peptide backbone and stub glycopeptide ions proved useful in assigningSWSYIAETPNSENGTCYPGYFADYEELR-[7,6,1,3,0] asthe correct com position
15. In case of AGP, SVQEIQATFFYFTPNK-[7,6,0,4,0]or QNQCFYNSSYLNVQRENGTVSR-[6,6,0,2,0] also correspondedto the same precursor mass 1088.2441 (5+), and SVQEIQATFFYFTPNK-[7,6,0,4,0]was assigned as the correct com position based on tandem MS data
16. Thefact that HILIC-C18-MS resulted in relative precursor ion abundancessufficient to allow selection for tandem MS and consequently makethese assignments demonstrates the value of this approach

*Output_Site_Fusion* (sent_index, protein, sugar, site):

Section : Confident Site-Specific Glycan Profiling

Content :

1. We used theglycopeptides assignments to construct a site-specific glycosylationmap for HA and AGP (Figures 4 and S-3, respectively)
2. Glycan com positions wereclassified, based on the number of HexNAc units, as high-mannose (2HexNAc) , biantennary (3 or 4 HexNAc) , triantennary (5 HexNAc) , tetra-antennary(6 HexNAc) , and penta-antennary (7 HexNAc)
3. Only glycopeptides identifiedwith at least 40% peptide backbone coverage from tandem MS data wereincluded in the glycan profile maps
4. In some cases, particularly forHA, two putative glycosylation sites appeared on a single trypticpeptide
5. The peptide backbone fragment ions with an attached HexNAchelped resolve the ambiguity in such cases by helping to identifythe occupied site
6. Such site-specific glycan profile assignments werepossible only when using HILIC-C18-MS/MS, whereby at least 7 timesmore glycopeptides underwent tandem-MS, from evaluation of precursorsgenerating oxonium ions
7. It is evident from the data that the usefulnessof HILIC-C18-MS scales with the number of glycosylation sites andglycopeptides in the theoretical search space
8. For transferrin , wherethe glycopeptide and glycoform diversity is very limited, the C18and HILIC-C18 platforms yield similar results
9. However, with AGP andhemagglutinin , there is an obvious improvement in data quality withthe use of HILIC-C18 over C18
10. We made these assignments using assumptionsregarding the protein sequences , glycosylation sites , glycoform distributions,and post-translational modifications
11. It is, therefore, possible thatmodified peptides not included in our assumptions exist
12. Since thegoal of this study was to compare performances of the HILIC-C18 andC18 systems, our glycopeptide analyses sufficed to reach clear conclusionsdespite the absence of exhaustive discovery-mode proteomics data interpretation

*Output_Site_Fusion* (sent_index, protein, sugar, site):

Section : Comparisonof tryptic glycopeptide abundances using (A) HILIC-C18-MSand (B) C18-MS

Content :

1. Each panel shows the base peak chromatogram (BPC)and the extracted ion chromatogram (EIC) for the HexNAc oxonium ion(m/z 204.08)
2. BPC and EIC are shown using the same y scale

*Output_Site_Fusion* (sent_index, protein, sugar, site):

Section : Comparisonof percent of precursor ions that generate diagnosticoxonium ions between HILIC-C18-MS (green) and C18-MS (red)

Content :

1. Resultsare shown for the glycoproteins labeled on the x axis

*Output_Site_Fusion* (sent_index, protein, sugar, site):

Section : Tandem mass spectrum of hemagglutinin glycopeptide precursor ion1013.7474 [M + 3H]3+, identified as NGSYPNLSK-[5,4,1,1,0]

Content :

1. Glycopeptide identifier is listed as peptide-[a ,b,c,d,e], where a= number of hexoses; b = number of N-acetylhexosamines;c = number of deoxyhexoses; d = number of N-acetylneuraminicacids; e = number of N-glycolylneuraminic acids

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Section : Site-specificglycan profile of hemagglutinin from influenza Avirus

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Protein	NCBI ID	SENTENCE INDEX