Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics. Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics. Protein glycosylation is a biologically significant and complex post-translational modification, involved in cell-cell and receptor-ligand interactions (1Jaeken J. Matthijs G. Congenital disorders of glycosylation.Annu. Rev. Genomics Hum. Genet. 2001; 2: 129-151Crossref PubMed Scopus (162) Google Scholar, 2Arnold J.N. Wormald M.R. Sim R.B. Rudd P.M. Dwek R.A. 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Here we on a IP-NPLC method using acids as ion-pairing reagents and as the for the isolation of tryptic The method and using a tryptic digest of B and fetuin mixture. In we that O-linked glycopeptides can be isolated from a fetuin tryptic digest by IP-NPLC of the N-linked by The IP-NPLC method used to N-linked glycopeptides from the tryptic digests of protein extracts of wild-type and PglD mutant of Campylobacter jejuni C. jejuni a that periplasmic and proteins containing the N-linked is J. L. N. E. of the N-linked glycan on multiple glycoproteins in the Campylobacter Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. D. M. M. A. M. N-linked glycosylation in Campylobacter jejuni and its E. PubMed Scopus Google Scholar, M. S. N. M. M. of the bacterial site J. 2006; 25: PubMed Scopus Google Scholar). The N-linked glycan of C. jejuni to be is J. L. N. E. of the N-linked glycan on multiple glycoproteins in the Campylobacter Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In the glycan structure of C. jejuni is in J. L. N. E. of the N-linked glycan on multiple glycoproteins in the Campylobacter Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. D. M. M. A. M. N-linked glycosylation in Campylobacter jejuni and its E. PubMed Scopus Google Scholar, M. S. N. M. M. of the bacterial site J. 2006; 25: PubMed Scopus Google Scholar). IP-NPLC close to 100% of the bacterial N-linked glycopeptides with of we for the first time that of bacillosamine is in the using IP-NPLC and MS. and the ion-pairing reagents from from The and the from The from from C. jejuni glycoprotein extracts from of as J. L. N. E. of the N-linked glycan on multiple glycoproteins in the Campylobacter Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). to a of using and B fetuin and with as W. J. of glycopeptides from glycoprotein digests using ion-pairing normal-phase 2007; PubMed Scopus Google Scholar). A of periplasmic protein extracts of C. jejuni 11168 the pglD mutant in with for and with for The reagents used for and removed by the the of of the protein with for the isolation a of to of of a fetuin tryptic digest with and for IP-NPLC on a to a The and IP-NPLC as The in of of ion-pairing in in with in R. C. M. E. between and in mixtures and 2007; PubMed Scopus Google Scholar). A of to B for to B for used a of The B for A is 100% The the that to the to for The of the to a The from the between are to as the the between are to as the The time to the elution time of the from of the B tryptic digest and used in IP-NPLC as a time for determination of the elution time of the IP-NPLC used in used for significant of The tryptic digest of periplasmic protein extracts of the C. jejuni 11168 and the pglD mutant of in of to IP-NPLC for isolation of The and for and to in of and by nano-flow using a to a The first a to a using a from to B in B for A in The from of of the glycopeptides isolated by IP-NPLC used for of periplasmic glycoproteins between the and mutant strains. with with a of for the digest and the and of the periplasmic protein of the pglD mutant of C. jejuni The with time of by and using to to and for determination of the glycan of the glycopeptides and for protein The first a to a using the as The of of the N-linked glycopeptides of C. jejuni proteins using and the C. jejuni with using the for protein The the by bacillosamine with a of J. L. N. E. of the N-linked glycan on multiple glycoproteins in the Campylobacter Biol. Full Text Full Text PDF PubMed Scopus Google and The of by used as of of C. jejuni that can be to The for is and the for is with The of and used for In to C. jejuni 11168 A tryptic digest of B and fetuin to the use of IP-NPLC for isolation The tryptic of B N-linked is and is with of the high mannose type to of and site in PubMed Scopus Google Scholar). N-linked and and O-linked and sialylated and of protein by reversed-phase and PubMed Scopus Google Scholar, A for identification and of glycoproteins using a 2006; PubMed Scopus Google Scholar). the non-glycopeptides of B and fetuin from of the of B and of the of The in to non-glycopeptides in of from to of non-glycopeptides calculated the using on the Wimley-White water/octanol for the acids a of and in a of 35: PubMed Scopus Google Scholar, K. measurement of using a for protein U. S. A. 93: PubMed Scopus Google Scholar, W. S. Scholar). the isolated glycopeptides and their from the tryptic digest using The in to the glycopeptides in of their calculated hydrophobicity values and IP-NPLC from the B and fetuin tryptic of hydrophobicity time of in a of glycopeptides and for the and O-linked from the B and fetuin tryptic of glycopeptides by of of the B and fetuin tryptic digest a in order to The of and are The glycopeptides by of of the B and fetuin tryptic digest a in order to The of and are of protein by reversed-phase and PubMed Scopus Google Scholar, A for identification and of glycoproteins using a 2006; PubMed Scopus Google Scholar). in a to the to the glycopeptides from B and the fetuin glycopeptides with MS from the the of fetuin of the of non-glycopeptides and the of to the non-glycopeptides and with the glycopeptides and the from of the In when to the the non-glycopeptides and the of the by from the B and fetuin digest and for of non-glycopeptides from the the a used in tryptic for non-glycopeptides and in the MS of the when to the the to the of glycopeptides and non-glycopeptides a ion-pairing used to in to the a of between glycopeptides and non-glycopeptides when with when the tryptic digest the to be to to the from non-glycopeptides A of glycopeptides with high mannose type glycopeptides from B sialylated N-linked glycopeptides and and sialylated O-linked glycopeptides from fetuin The of the B and fetuin tryptic digest reproducible on on the of and with of high mannose type glycopeptides close to 100% The of acids on time of a and that of a is acids to the and for the the the with the in of and B and In when the with time differences of between the and of from the time for IP-NPLC on a and using the as for the A that a of used for the The are in that the A is the is and the is The of to the isolation of isolation considerably as the and The A to the The that interactions between and the are to The A and a on its from in IP-NPLC using We the calculated hydrophobicity values of the tryptic non-glycopeptides of B and fetuin their IP-NPLC elution with the of to the The of the and the of of IP-NPLC that of a with glycopeptides in IP-NPLC can be using their calculated hydrophobicity the of B a hydrophobicity of and the tryptic non-glycopeptides in the digest and In the from non-glycopeptides with hydrophobicity values are to glycopeptides modified by high mannose type heptasaccharides in The of tryptic of proteins hydrophobicity values is that IP-NPLC is of glycopeptides from tryptic digests of complex protein mixtures. The O-linked from a fetuin tryptic digest by the MS of these O-linked with of the N-linked of the glycosylation A for identification and of glycoproteins using a 2006; PubMed Scopus Google Scholar). the fetuin tryptic digest with to the N-linked the in the IP-NPLC from O-linked glycopeptides of fetuin The is of a complex tryptic digest of periplasmic protein extracts of the C. jejuni 11168 and the pglD mutant isolated by IP-NPLC and to The of the and IP-NPLC and the digest are in The of the and digest and that of the complex of of non-glycopeptides by that the from the of the from glycopeptides the of the and of glycoproteins from the periplasmic protein extracts of C. jejuni 11168 and the pglD and of for of are in of periplasmic is by and glycoproteins are is monoacetylated the The of for proteins are the as are for on and used for with periplasmic periplasmic protein to periplasmic periplasmic periplasmic The and of for of are in The periplasmic is by and glycoproteins are is monoacetylated the J. 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S. periplasmic protein periplasmic in in protein periplasmic A. of in periplasmic in periplasmic in in in a The in the of the and pglD mutant extracts to and to their and their The N-linked glycan of C. jejuni to be is J. L. N. E. of the N-linked glycan on multiple glycoproteins in the Campylobacter Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In in PglD as the acetyltransferase that the the to using A as the B In of by of the Campylobacter jejuni protein glycosylation 2006; PubMed Scopus Google Scholar). the of glycopeptides from the and mutant are in a and are by with of from the glycan the of the to be The of the pglD is to that of the that and order are in the strains. the is by in the mutant and as The glycan and structure of are as on the to the the to from the C. jejuni periplasmic with of as by the A of glycopeptides to periplasmic glycoproteins by for and J. L. N. E. of the N-linked glycan on multiple glycoproteins in the Campylobacter Biol. 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M. of the bacterial site J. 2006; 25: PubMed Scopus Google and glycopeptides from and The and for these glycopeptides are in The for from the pglD digest as as the and IP-NPLC are in The in the and and and in the and The in the and for of the glycopeptides in of their that the of these glycopeptides by IP-NPLC close to a from the a the of as non-glycopeptides that glycopeptides in the and of the of the pglD mutant using and of the The in as the and their are in The in for the of the of the of the relative to the for The of the as the of glycopeptides by the the of glycopeptides by the of the from the in and in in the from the of these in a complex tryptic digest the of In we describe a IP-NPLC method for glycopeptides from complex tryptic digests using acids as ion-pairing reagents and A The of acids to the to of non-glycopeptides with glycopeptides from the B and fetuin tryptic digest and In the use of in of glycopeptides from non-glycopeptides and is that the in isolation of glycopeptides using IP-NPLC of the in the to isolation of glycopeptides the and in the can the of on isolation can be by the of the acids and The that in the the of for the isolation of A of sialylated glycopeptides from O-linked and N-linked glycopeptides, and high mannose type N-linked glycopeptides from B isolated by IP-NPLC from a tryptic digest of a of these glycoproteins and of protein by reversed-phase and PubMed Scopus Google Scholar, A for identification and of glycoproteins using a 2006; PubMed Scopus Google Scholar, P. 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We for the first time that the of the isolation using the elution order of non-glycopeptides in IP-NPLC can be using their hydrophobicity values with a hydrophobicity will in the In we that IP-NPLC can the O-linked glycopeptides from a fetuin digest the N-glycans removed with This and we to to the of in biological We IP-NPLC to and glycopeptides from tryptic digests of periplasmic proteins from C. jejuni 11168 and pglD mutant for of glycoprotein We to the C. jejuni pglD mutant with in H. L. L. B. of the N-linked glycan in Campylobacter jejuni and protein 2006; PubMed Scopus Google we of protein glycosylation in mutant of to these of the glycopeptides in the isolated from the is from the periplasmic This glycoprotein a of and by the used in to the glycoprotein mixtures to MS J. L. N. E. of the N-linked glycan on multiple glycoproteins in the Campylobacter Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the of MS using IP-NPLC is with that using and H. R. and quantification of N-linked glycoproteins using and PubMed Scopus Google Scholar). In IP-NPLC as for and the glycan The of monoacetylated on glycoproteins isolated from the jejuni pglD mutant confirms that PglD is acetyltransferase and that the is of the quantitative on the differentially expressed N-linked glycoproteins further demonstrates the of for activity in C. jejuni M. N. M. M. D. A. C. M. of bacterial a for the bacterial and U. S. A. 2006; PubMed Scopus Google Scholar). The quantitative for the first time that glycopeptides modified by are in the that of is in the of PglD In is that is acetyltransferase the PglD activity as we H. L. L. B. of the N-linked glycan in Campylobacter jejuni and protein 2006; PubMed Scopus Google glycopeptides modified by are in the mutant with the The of on the of glycopeptides and in the C. jejuni and on the time between the and the glycopeptides, in the B and fetuin digest IP-NPLC glycopeptides modified with a to to be from complex tryptic the be of glycopeptides by IP-NPLC is close to 100% for the on the of in the This and is with that with tryptic glycopeptides B. X. B. L. with for 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). We believe that IP-NPLC method can be used for glycopeptides glycopeptides from the tryptic digests of periplasmic protein extracts from C. jejuni In to for glycopeptides, IP-NPLC method the and for complex mixtures to J. D. using glycoproteins with to cancer 2006; PubMed Scopus Google Scholar, M.R. P. P. of glycoproteins using proteolytic with and 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. P. 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