Large-scale Identification of N-linked Intact Glycopeptides in Human Serum using HILIC Enrichment and Spectral Library Search

Abstract

Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both N-linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level. Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both N-linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level. N-linked glycoproteins in human serum have been utilized in diagnosing of diseases for decades, such as prostate specific antigen (PSA) 1The abbreviations used are:PSAprostate specific antigenACNacetonitrileCA-125cancer antigen 125CDcluster of differentiationCDGcongenital disorders of glycosylationCDTcarbohydrate-deficient transferrinCFHcomplement factor HDDAdata-dependent acquisitionDIAdata-independent acquisitionDTT1, 4-dithiothreitolFAformic acidFDGPfractionated deglycopeptidesFGPfractionated glycopeptidesFDRfalse discovery rateGAGglycosaminoglycanGPSMglycopeptidespectrum matchHILIChydrophilic interaction liquid chromatographyIAM2-iodoacetamideLCliquid chromatographyLC-MS/MSliquid chromatography coupled tandem mass spectrometryMSmass from from liquid from from from serum from serum 1The abbreviations used are:PSAprostate specific antigenACNacetonitrileCA-125cancer antigen 125CDcluster of differentiationCDGcongenital disorders of glycosylationCDTcarbohydrate-deficient transferrinCFHcomplement factor HDDAdata-dependent acquisitionDIAdata-independent acquisitionDTT1, 4-dithiothreitolFAformic acidFDGPfractionated deglycopeptidesFGPfractionated glycopeptidesFDRfalse discovery rateGAGglycosaminoglycanGPSMglycopeptidespectrum matchHILIChydrophilic interaction liquid chromatographyIAM2-iodoacetamideLCliquid chromatographyLC-MS/MSliquid chromatography coupled tandem mass spectrometryMSmass from from liquid from from from serum from serum and antigen and glycoproteins as specific for identification of N-linked intact glycopeptides is a to under LC-MS/MS been to and N-linked glycopeptides. In this is In for to a as the of and in this at the a of by the the most of this in A of to spectra of of data by mass the to and the most such as for and by the the is for during and as the this the the of N-linked intact glycopeptide is the most is as in to a recognize the of N-linked intact glycopeptides is in the In with of to separated by used liquid chromatography of N-linked intact glycopeptides in and of in in of glycopeptides. of and to of of N-linked intact glycopeptides in spectra under the of a was in a data from to of protein glycosylation by of N-linked and In a with spectral library search was utilized in N-linked intact glycopeptide identification from human of of to the and a method for of in mass was into and was to N-linked intact glycopeptide identification database search with and mass spectrometry for intact glycopeptide Both in-depth identification of N-linked intact glycopeptides in were in identification from the of and human serum a dynamic range in protein is a to the N-linked intact glycopeptides in human the spectral library of N-linked deglycopeptides to glycoproteins and of in at by was by of proteins and of N-linked intact by of F to generate N-linked deglycopeptides and LC-MS/MS of the of to in this is to of N-linked intact glycopeptide false N-linked glycosites by of the false identification of glycosites, a was to N-linked glycosites and in of were with N-linked were by F digestion, and the N-linked deglycopeptides were to by of protein glycosylation by of N-linked and with the this was to human serum and N-linked glycoproteins were identified of Serum and and the and serum the of serum N-glycoproteome by this was prostate specific antigen acetonitrile antigen of disorders of glycosylation factor deglycopeptides glycopeptides false discovery rate hydrophilic interaction liquid chromatography liquid chromatography liquid chromatography coupled tandem mass spectrometry mass spectrometry deglycopeptides from glycopeptides from liquid chromatography deglycopeptides from glycopeptides from deglycopeptides from serum glycopeptides from serum prostate specific antigen acetonitrile antigen of disorders of glycosylation factor deglycopeptides glycopeptides false discovery rate hydrophilic interaction liquid chromatography liquid chromatography liquid chromatography coupled tandem mass spectrometry mass spectrometry deglycopeptides from glycopeptides from liquid chromatography deglycopeptides from glycopeptides from deglycopeptides from serum glycopeptides from serum In this a complete to the of serum N-glycoproteome LC-MS/MS. serum proteins were separated into low-abundance and high-abundance by precipitation. After digestion, was used to in glycopeptide of Serum and and of for in of F was used to N-linked deglycopeptides from a portion of the enriched N-linked intact glycopeptide The N-linked deglycopeptides were analyzed LC-MS/MS and identified protein database search a for and protein identification tandem mass From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. The of serum N-glycoproteome identified by this was to A spectral library of identified deglycopeptides with was by N-linked glycopeptide identification mass spectral library N-linked intact glycopeptides were analyzed LC-MS/MS. of N-linked intact glycopeptides was used for N-linked intact glycopeptide was to the of N-linked intact glycopeptides. spectral library search and target-decoy 526 N-linked glycoproteins were identified in serum 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at intact N-linked glycopeptide and N-linked level. In this serum from and serum from were A of for of human serum is in the serum and portion of enriched N-linked intact glycopeptide was analyzed by LC-MS/MS for and N-linked intact glycopeptides N-linked intact portion of enriched N-linked intact glycopeptide was with F and analyzed by LC-MS/MS for and N-linked deglycopeptides N-linked serum proteins were separated into and protein in the and the by precipitation. in the and the were processed with and to generate N-linked intact glycopeptide portion of enriched N-linked intact glycopeptides was into by and analyzed by portion of enriched N-linked intact glycopeptide was with into by and analyzed by LC-MS/MS The LC-MS/MS data sets, N-linked intact glycopeptides and N-linked deglycopeptides with N-linked intact glycopeptides from and N-linked intact glycopeptides from with N-linked deglycopeptides from and deglycopeptides from The for the LC-MS/MS with for the is the of N-linked deglycopeptides identified LC-MS/MS is and the of N-linked deglycopeptides identified of in N-glycoproteome and the to the by and of identified N-linked intact glycopeptides LC-MS/MS is because a and serum a LC-MS/MS by for from the by from a a used to serum for for of to serum for Four and were in of these of serum of of the glycoproteins, is in a from was in this to the of N-linked of serum to and by LC-MS/MS in a from and the of N-linked glycoproteins, glycosites, intact and identified in the and and the of in the of serum N-linked of of N-linked in serum is in this method into and data is in the of this was and by the at of of Serum were from in at the of the were for were with and at for of serum from were used for glycopeptide and identification of from were used for from was from and human were from and were processed the used to generate N-linked deglycopeptides and N-linked intact glycopeptides. intact glycopeptides and intact glycopeptides were with human serum intact glycopeptides. The was into portion was with F to generate the N-linked deglycopeptides by LC-MS/MS with and portion was analyzed by LC-MS/MS with In the of serum and at for was at at for The and were and by The serum protein and were in by by at for and by at in the for The was by at for The was with a of the was in and at with was by to a of The was with The were with and The were and to by was by of Serum N-linked intact glycopeptides were enriched by a method and of and in protein and The of N-linked intact glycopeptides was a with a and a The of N-linked intact glycopeptides from of in this was the method as of was and by in by for The was with for was the a and were with A in and in at a and at a of The was at The N-linked intact glycopeptides containing were from to to the and by After of by the of glycopeptide were to for to the of After portion of N-linked intact glycopeptide was in for LC-MS/MS portion was with F to generate deglycopeptides. of the enriched N-linked intact glycopeptides from serum of F was into of N-linked intact glycopeptide in and at to After F the were by with and with The were and by and in for LC-MS/MS of enriched N-linked intact glycopeptides from of F was into of N-linked intact glycopeptide in and at to After F the were by with and with The were and by and in for LC-MS/MS The of enriched N-linked intact glycopeptide and were a with a a and a The of N-linked intact glycopeptides and deglycopeptides in this were the method as The enriched N-linked intact glycopeptides deglycopeptides from and were in into a with A and at a and at a of The was at of N-linked intact glycopeptides deglycopeptides were in a method from to and into by The were by and at LC-MS/MS of deglycopeptides from human and glycoproteins and serum with and were coupled with mass were in and into a and separated a with A and at a and at a of The mass was in spectra range from to were in the with of at The was as and the was as The were from with of were in the with of spectra were in the with of at The was and the was for were for a of The of were as and the was was The was and the was LC-MS/MS of were the method with of the mass range to the N-linked intact glycopeptides from human and the with and LC-MS/MS were the method with of the mass range to and to The of were as and the was and was LC-MS/MS spectra of N-linked deglycopeptides in and as as the with and were to with the as After and spectra were the protein database from of by The search were as to mass and mass was a and were the search by the of the spectra were by the and as with and with and with and with and with and with with and identification of N-linked deglycopeptides from the spectra were the protein database of in from of with and were as these in search of into because the of data was identification of N-linked deglycopeptides from the spectra were the protein database of in with and were as identification of N-linked deglycopeptides of the spectra were the protein database of in The spectra were by with and with and and identification of N-linked deglycopeptides of serum with and the spectra were the protein database of with proteins from and proteins from The was the as for were as for search identified have were with 1% at the by the of identified N-linked deglycopeptides were for N-glycan were from database for in and masses with and the masses from N-glycan database with N-glycan masses with and from been used in the of with and mass spectrometry for intact glycopeptide the with and N-glycan masses were to a 739 N-glycan masses and was of of and and The of and N-glycan masses used in is N-glycan masses with The of N-glycan masses and The N-linked glycopeptide identification mass spectral library was used to N-linked intact glycopeptides. data from N-linked intact glycopeptides were from to with the and with the A spectral library of N-linked deglycopeptides was constructed from identified N-linked deglycopeptides in and data The identified N-linked deglycopeptides were with the of the N-linked glycosylation and with a X≠P) the with of this during The spectral library of N-linked deglycopeptides was constructed by to of spectra of the identified N-linked deglycopeptides. mass and mass were used during library was as was as of were as The spectra were mass was used during library and mass and mass were used during of N-linked intact glycopeptides. N-linked deglycopeptides identified in and and were for serum N-linked deglycopeptides library The of of was as and for and data sets, N-glycan mass containing and 739 N-glycan masses were a spectral library was identified N-linked deglycopeptides. A database with N-glycan masses from as of The of was used for N-linked intact glycopeptides. A database with N-glycan masses from was used for N-linked intact glycopeptides. A database with 739 N-glycan masses was used for N-linked intact glycopeptides. of were as of was as The N-linked were The spectra were from the and data the target-decoy to the of N-linked intact glycopeptide in the library construction for a was with the mass and The was by the the of by the N-linked intact glycopeptide was the was with to the by and were were by and the was the and the of to the and search for in protein by mass the glycopeptide were separated into to motifs and 1% was the of in was for by the the of were for the target-decoy was false discovery rate for by mass After the 1% false discovery rate for by mass was as a to The were the a was as a by In this a was by to have and was identified by as under 1% the to the in the of the were in to the these from were in is the the was the was and the the was the was for these identified from were and as N-linked intact were into of The of a glycopeptide as as to were used to with a of mass were the in A by the was used to the The was by the of a glycopeptide into a of a was from generate used from with as the mass and masses were to have at mass from of the N-glycan masses to from the in the mass the of this a of glycoproteins and were into serum and the of identified N-glycan masses were The data and protein database search for N-linked database the of serum to in to the of N-linked was as because the of proteins in human is for spectra from of LC-MS/MS the human and is to in the The of mass search this to The is in such as and the in human serum used the and of protein in search and to in human to the in the data mass From this from types of mass were and and of these in serum N-linked deglycopeptides to was because was used during this of with a protein N-linked deglycopeptides with this were identified in and data and of identified N-linked deglycopeptides were in and is to in database search for the identification of serum N-linked deglycopeptides. 1% FDR, and N-linked deglycopeptides to and N-linked glycosites from and N-linked glycoproteins were identified in and The of N-linked glycoproteins, glycosites and deglycopeptides were by and with the of and the and protein and N-linked the of serum A of 3328 N-linked deglycopeptides have been identified in human the from and were from 764 N-linked glycoproteins 1699 N-linked The of serum N-glycoproteome identified by this was to of from N-linked of by in a is a to a complete N-glycan database for identification of N-glycoproteome by of were from in a database with N-glycan masses the masses from In this N-glycan mass of masses were from containing and such as and were as and the masses were were of of and N-glycan database with with human and from been used in the of with and mass spectrometry for intact glycopeptide by from human and The N-glycan masses with N-glycan and were in this for mass spectral library search was in to from the N-glycan mass the mass a and a library a was this and 1% target-decoy the of 1% were to motifs motifs (NXS/T/C/V, X≠P) in 1% was After 1% a of identified glycopeptide by were to because were the and masses by these glycopeptides have and from In this the mass of by masses with mass these from and the unique The of these by from is is the of is and is by the high-abundance the of identified by from have the were to the with the is the the is the N-linked glycosites and N-linked intact glycopeptides from N-linked glycoproteins were identified from and data the database of N-glycan masses and In N-linked glycosites and N-linked intact glycopeptides from N-linked glycoproteins were identified the database of N-glycan masses and N-glycan masses were identified and N-glycan masses in and data The N-glycan masses and to N-glycan masses and were and of at and the constructed N-linked library most of the serum N-linked The of N-linked intact glycopeptides by N-linked masses database to N-glycan masses the of human in database is A human is to the in-depth of human serum N-linked The and N-glycan masses were The database 739 N-glycan masses and was used to the serum data and In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were the most in-depth N-glycoproteome in serum identified by LC-MS/MS at N-linked intact glycopeptide and to the the N-glycan the of by and at the and glycopeptide the mass database and of from N-linked intact glycopeptide N-glycan by in a of from N-linked intact glycopeptide N-glycan by in a of from N-linked glycopeptide 739 N-glycan by in a the of N-glycan in serum glycoproteins, as by and the proteins with the most glycosites identified in and and glycosites were identified from In the intact glycopeptide data and and glycosites, and and were identified from and The of 739 N-glycan masses used for serum N-linked intact glycopeptide identification is in is to a The of mass in the range of the 739 masses is in and the the of mass in the range of is The of and the of of identification the identification of N-glycan of the and the was used for masses identified in and the 739 N-glycan masses and 739 N-glycan masses were for The identified N-glycan masses to and in and In N-glycan masses were identified in and the N-glycan and to the most by human serum In these the and the of in the serum data After the masses with the masses and the serum data sets, and of in and to the masses the of identification was of the masses this masses were with In this the rate is the masses as a to and the of used this as method to the identification used as as the serum with these proteins and to identification The serum was used as a as was identified with of in the human serum data From the protein identified glycosites and of masses is to the in N-linked intact glycopeptides in protein identified proteins in the protein is proteins were identified from the the glycoproteins and and most of the identified from this were from of the in a with The and the glycoproteins and were used to the of database search N-glycan masses from and N-glycan masses from in were used to N-linked intact glycopeptides. Four of search were and and the most from the proteins the glycosites of and were identified by to the of and the of and N-glycan masses identified from of of these by were the of and in the the of this a of glycoproteins and into serum and analyzed the of identified N-glycan After into the of identified N-glycan masses by and the N-linked glycosites and of the of identified N-glycan masses by and the N-linked glycosites of the of identified N-glycan masses by and the N-linked glycosites and of the of identified N-glycan masses in of was identified into serum and the of identified N-glycan masses in the of this of the is by the intact spectra from a the protein and mass database from the N-linked intact glycopeptide data from human serum N-linked library and the 739 N-glycan masses were to the the N-linked library and N-glycan masses database from the of the was as the N-glycan is to processed by F and of a with of F and of proteins in processed by F to by the N-linked intact glycopeptides this N-glycan in identified the N-linked deglycopeptides processed by identified this glycosites and in The identified 526 serum were to the diseases and and the of enriched by the database The database in and was the most enriched by The and as the enriched The the of by and to The and a of human most in this in The most enriched of the 526 serum glycoproteins is and the is factor is a to the of in by to interaction with factor and and in from were identified in this the of identified of was the and to and the of the in the protein is is to The of and with to for the of and and were enriched in the identified serum glycoproteins of these proteins and from such as from the from A is a of and of The of at glycopeptide containing this were identified the 739 N-glycan is of in the serum of is a of and in of with and the to is a for and is to in serum from into during The of these of The method the by a complete database of as as N-linked deglycopeptides from human this database is to in serum was in a of with and In serum of were used for N-linked glycopeptide identification of Serum and and the of of mass the of mass with N-glycan masses and the with N-glycan The the of N-glycan mass database the of N-linked intact glycopeptides. the of N-linked N-linked was of N-glycan masses with in the N-glycan masses were to for A method to as for The mass were and used to the serum N-linked intact glycopeptide data In total, serum N-linked glycoproteins with N-linked glycosites were identified to these with the identified the of these were identified the of and in serum proteins such as and of identified in the is to serum N-linked intact glycopeptides. in N-glycan of is is to in a The identified N-linked intact glycopeptides 739 N-glycan masses database were used to for a and factor were as were N-linked glycosites of and The of glycosites of serum with glycoproteins, and data The of and of factor serum with glycoproteins, and data the glycosites of and in Both A and were by of at protein the glycosites and were to at the of and protein A for by The N-glycan and and N-glycan masses were identified the N-glycan masses were identified The and The was identified as by was to The is from the the of N-glycan masses identified. In this regard, the of N-glycan mass identified the of the of to and From the of is to a of of in in to and human proteins in the of and the of identified in to in protein A of in is to a the in a After by to and to by to is a is used in of types with in the identification of in N-glycan is by the of and disorders of by of in a because the intact protein is into the of these identified the of the of was by for and N-glycan masses were identified with the at of A of this was in In total, N-glycan masses were identified of from and data In and the to a a identified LC-MS/MS by the of in is a to identified in and was to the of a glycopeptide the in was at in the of this glycopeptide in LC-MS/MS was by the of the The of this N-linked intact glycopeptide was by is of in LC-MS/MS for the of of this was with was by a and in used with of to the of in N-linked intact glycopeptide a was and as a spectral library for of glycosylation in human serum to the of N-linked intact glycopeptides containing the such as of from and of the of glycopeptide the and is and the of this in a to recognize glycopeptide and is in this with for N-linked intact glycopeptide the of proteins in serum been into in the and were during database the of these have been in as the used a of to the of digestion, and the the of of these protein glycopeptides. In the of was to in to N-linked deglycopeptides. In of were the glycosites and N-glycan masses from these and the human serum data under of search were by and by false the database search from and from were as The were with 1% at the identified as and the were used to serum N-linked spectral In this regard, the of was and the with of the and and in the of because of the lack of containing the is to the in N-linked intact glycopeptide the have as the of false by of during data was masses into search the of method was The of and masses in the is a of data to recognize N-glycan masses and glycopeptides is in the human serum N-glycan method was used to at the for In the is the mass In the the target-decoy was for of of the of the and of a is the mass of the mass of the N-linked intact glycopeptide is to identification of the of a the identified the because and in database The mass by these as of a to identification of the have and into database the of N-linked intact glycopeptides with is to and the identification by this is the mass is and the mass the in the of the and to a mass to the the rate of mass by the masses and into the database for because of the of masses and the is the mass the of mass identification was to to the of human serum this is because is to mass into the masses were used for method of control. at the were in human serum of Serum and and is in to false by this identified is by database search in N-linked intact glycopeptide identification to this in N-linked intact glycopeptide identification and to in In the and data sets, identified deglycopeptides with into the spectral N-linked glycoproteins N-linked glycosites were identified with the of and of of Serum and and method was is this identified is from the motifs in of and by is in the and of disorders and to disorders of glycosylation serum glycoproteins in the method used for and is used as the to because of of disorders of glycosylation by of In the serum of with the of from was identified by In this is identified the glycosites of with the and method and of in a the identified a spectral library of glycopeptide constructed and to for to of of human and the of protein to and from The the of for a N-glycan the in with the of in the of the of in by in-depth of and the of to identification of N-glycan is a for of of serum The LC-MS/MS method for N-linked glycopeptide identification and construction was in human serum of of serum to for and of as as human The mass spectrometry data and data from glycoproteins as as serum with and and N-linked intact glycopeptide spectra have been to the the of the database and with the data The data was of protein glycosylation by of N-linked and and with the data The and for and at for during data and for in and data and for in and data and and for the and for with

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