25 research outputs found
Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry
Glycosylation is one of the most common post-translational modifications in proteins, existing in âŒ50% of mammalian proteins. Several research groups have demonstrated that mass spectrometry is an efficient technique for glycopeptide identification; however, this problem is still challenging because of the enormous diversity of glycan structures and the microheterogeneity of glycans. In addition, a glycopeptide may contain multiple glycosylation sites, making the problem complex. Current software tools often fail to identify glycopeptides with multiple glycosylation sites, and hence we present GlycoMID, a graph-based spectral alignment algorithm that can identify glycopeptides with multiple hydroxylysine O-glycosylation sites by tandem mass spectra. GlycoMID was tested on mass spectrometry data sets of the bovine collagen α-(II) chain protein, and experimental results showed that it identified more glycopeptide-spectrum matches than other existing tools, including many glycopeptides with two glycosylation sites
LCâMS/MS Identification of the OâGlycosylation and Hydroxylation of Amino Acid Residues of Collagen αâ1 (II) chain from Bovine Cartilage
O-Glycosylation
of collagen is a unique type of posttranslational
modifications (PTMs) involving the attachment of galactose (Gal) or
glucose-galactose (Glc-Gal) moieties to hydroxylysine (HyK). Also,
hydroxyproline (HyP) result from the posttranslational hydroxylation
of some proline residues in collagen. Here, LCâMS/MS was effectively
employed to identify 23 O-glycosylation sites and a large number of
HyP residues associated with bovine type II collagen α-1 chain
(CO2A1). The modifications of the 23 O-glycosylation sites varied
qualitatively and quantitatively. Both Gal and Glc-Gal moieties occupied
22 of the identified glycosylation sites, while K773 was observed
as unmodified. A large number of HyP residues at Yaa positions of
Gly-Xaa-Yaa motif were detected. HyP residues at Xaa positions of
Gly-HyP-HyP, Gly-HyP-Ala, and Gly-HyP-Val motifs were also observed.
Notably, HyP residue of Gly-HyP-Gln motif was detected, which has
not been previously reported. Moreover, the deamidation of 8 Asn residues
was identified, of which 2 Asp residues were observed at different
retention times because of isomerization (Asp vs isoAsp). Partial
macroheterogeneities of some CO2A1 glycosylation sites were revealed
by LCâMS/MS analysis. ETD experiments revealed partial macroheterogeneities
associated with K299âK308, K452âK464, K464âK470,
and K857âK884 glycosylation sites. Semiquantitative data suggest
that the glycosylation of hydroxylysine residues is site-specific
LCâMS/MS Identification of the OâGlycosylation and Hydroxylation of Amino Acid Residues of Collagen αâ1 (II) chain from Bovine Cartilage
O-Glycosylation
of collagen is a unique type of posttranslational
modifications (PTMs) involving the attachment of galactose (Gal) or
glucose-galactose (Glc-Gal) moieties to hydroxylysine (HyK). Also,
hydroxyproline (HyP) result from the posttranslational hydroxylation
of some proline residues in collagen. Here, LCâMS/MS was effectively
employed to identify 23 O-glycosylation sites and a large number of
HyP residues associated with bovine type II collagen α-1 chain
(CO2A1). The modifications of the 23 O-glycosylation sites varied
qualitatively and quantitatively. Both Gal and Glc-Gal moieties occupied
22 of the identified glycosylation sites, while K773 was observed
as unmodified. A large number of HyP residues at Yaa positions of
Gly-Xaa-Yaa motif were detected. HyP residues at Xaa positions of
Gly-HyP-HyP, Gly-HyP-Ala, and Gly-HyP-Val motifs were also observed.
Notably, HyP residue of Gly-HyP-Gln motif was detected, which has
not been previously reported. Moreover, the deamidation of 8 Asn residues
was identified, of which 2 Asp residues were observed at different
retention times because of isomerization (Asp vs isoAsp). Partial
macroheterogeneities of some CO2A1 glycosylation sites were revealed
by LCâMS/MS analysis. ETD experiments revealed partial macroheterogeneities
associated with K299âK308, K452âK464, K464âK470,
and K857âK884 glycosylation sites. Semiquantitative data suggest
that the glycosylation of hydroxylysine residues is site-specific
LCâMS/MS Identification of the OâGlycosylation and Hydroxylation of Amino Acid Residues of Collagen αâ1 (II) chain from Bovine Cartilage
O-Glycosylation
of collagen is a unique type of posttranslational
modifications (PTMs) involving the attachment of galactose (Gal) or
glucose-galactose (Glc-Gal) moieties to hydroxylysine (HyK). Also,
hydroxyproline (HyP) result from the posttranslational hydroxylation
of some proline residues in collagen. Here, LCâMS/MS was effectively
employed to identify 23 O-glycosylation sites and a large number of
HyP residues associated with bovine type II collagen α-1 chain
(CO2A1). The modifications of the 23 O-glycosylation sites varied
qualitatively and quantitatively. Both Gal and Glc-Gal moieties occupied
22 of the identified glycosylation sites, while K773 was observed
as unmodified. A large number of HyP residues at Yaa positions of
Gly-Xaa-Yaa motif were detected. HyP residues at Xaa positions of
Gly-HyP-HyP, Gly-HyP-Ala, and Gly-HyP-Val motifs were also observed.
Notably, HyP residue of Gly-HyP-Gln motif was detected, which has
not been previously reported. Moreover, the deamidation of 8 Asn residues
was identified, of which 2 Asp residues were observed at different
retention times because of isomerization (Asp vs isoAsp). Partial
macroheterogeneities of some CO2A1 glycosylation sites were revealed
by LCâMS/MS analysis. ETD experiments revealed partial macroheterogeneities
associated with K299âK308, K452âK464, K464âK470,
and K857âK884 glycosylation sites. Semiquantitative data suggest
that the glycosylation of hydroxylysine residues is site-specific
Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment
LCâMS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography
Changes
in glycosylation have been shown to have a profound correlation
with development/malignancy in many cancer types. Currently, two major
enrichment techniques have been widely applied in glycoproteomics,
namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry
(HC)-based enrichments. Here we report the LCâMS/MS quantitative
analyses of human blood serum glycoproteins and glycopeptides associated
with esophageal diseases by LAC- and HC-based enrichment. The separate
and complementary qualitative and quantitative data analyses of protein
glycosylation were performed using both enrichment techniques. Chemometric
and statistical evaluations, PCA plots, or ANOVA test, respectively,
were employed to determine and confirm candidate cancer-associated
glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins
(42% overlap) were observed in both enrichment techniques. This overlap
is very similar to previously published studies. The quantitation
and evaluation of significantly changed glycoproteins/glycopeptides
are complementary between LAC and HC enrichments. LCâESIâMS/MS
analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins
enriched by HC showed significantly different abundances between disease-free
and disease cohorts. Multiple reaction monitoring quantitation resulted
in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by
HC enrichment to be statistically different among disease cohorts
Glycoproteomics: Identifying the Glycosylation of Prostate Specific Antigen at Normal and High Isoelectric Points by LCâMS/MS
Prostate
specific antigen (PSA) is currently used as a biomarker
to diagnose prostate cancer. PSA testing has been widely used to detect
and screen prostate cancer. However, in the diagnostic gray zone,
the PSA test does not clearly distinguish between benign prostate
hypertrophy and prostate cancer due to their overlap. To develop more
specific and sensitive candidate biomarkers for prostate cancer, an
in-depth understanding of the biochemical characteristics of PSA (such
as glycosylation) is needed. PSA has a single glycosylation site at
Asn69, with glycans constituting approximately 8% of the protein by
weight. Here, we report the comprehensive identification and quantitation
of N-glycans from two PSA isoforms using LCâMS/MS. There were
56 N-glycans associated with PSA, whereas 57 N-glycans were observed
in the case of the PSA-high isoelectric point (pI) isoform (PSAH).
Three sulfated/phosphorylated glycopeptides were detected, the identification
of which was supported by tandem MS data. One of these sulfated/phosphorylated
N-glycans, HexNAc5Hex4dHex1s/p1 was identified in both PSA and PSAH
at relative intensities of 0.52 and 0.28%, respectively. Quantitatively,
the variations were monitored between these two isoforms. Because
we were one of the laboratories participating in the 2012 ABRF Glycoprotein
Research Group (gPRG) study, those results were compared to that presented
in this study. Our qualitative and quantitative results summarized
here were comparable to those that were summarized in the interlaboratory
study
Characterization of the Glycosylation Site of Human PSA Prompted by Missense Mutation using LCâMS/MS
Prostate
specific antigen (PSA) is currently used as a diagnostic
biomarker for prostate cancer. It is a glycoprotein possessing a single
glycosylation site at N69. During our previous study of PSA N69 glycosylation,
additional glycopeptides were observed in the PSA sample that were
not previously reported and did not match glycopeptides of impure
glycoproteins existing in the sample. This extra glycosylation site
of PSA is associated with a mutation in KLK3 genes. Among single nucleotide
polymorphisms (SNPs) of KLKs families, the rs61752561 in KLK3 genes
is an unusual missense mutation resulting in the conversion of D102
to N in PSA amino acid sequence. Accordingly, a new N-linked glycosylation
site is created with an N102MS motif. Here we report the first qualitative
and quantitative glycoproteomic study of PSA N102 glycosylation site
by LCâMS/MS. We successfully applied tandem MS to verify the
amino acid sequence possessing N102 glycosylation site and associated
glycoforms of PSA samples acquired from different suppliers. Among
the three PSA samples, HexNAc2Hex5 was the predominant glycoform at
N102, while HexÂNAc4ÂHex5ÂFuc1ÂNeuÂAc1 or
HexÂNAc4ÂHex5ÂFuc1ÂNeuÂAc2 was the primary
glycoforms at N69. D102 is the first amino acid of âkallikrein
loopâ, which is close to a zinc-binding site and catalytic
triad. The different glycosylation of N102 relative to N69 might be
influenced by the close vicinity of N102 to these functional sites
and steric hindrance
Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry
Computational Framework for Identification of Intact Glycopeptides in Complex Samples
Glycosylation is an important protein
modification that involves
enzymatic attachment of sugars to amino acid residues. Understanding
the structure of these sugars and the effects of glycosylation are
vital for developing indicators of disease development and progression.
Although computational methods based on mass spectrometric data have
proven to be effective in monitoring changes in the glycome, developing
such methods for the glycoproteome are challenging, largely due to
the inherent complexity in simultaneously studying glycan structures
with their corresponding glycosylation sites. This paper introduces
a computational framework for identifying intact N-linked glycopeptides,
i.e. glycopeptides with N-linked glycans attached to their glycosylation
sites, in complex proteome samples. Scoring algorithms are presented
for tandem mass spectra of glycopeptides resulting from collision-induced
dissociation (CID), higher-energy C-trap dissociation (HCD), and electron
transfer dissociation (ETD) fragmentation modes. An empirical false-discovery
rate estimation method, based on a target-decoy search approach, is
derived for assigning confidence. The power of our method is further
enhanced when multiple data sets are pooled together to increase identification
confidence. Using this framework, 103 highly confident N-linked glycopeptides
from 53 sites across 33 glycoproteins were identified in complex human
serum proteome samples using conventional proteomic platforms with
standard depletion of the 7-most abundant proteins. These results
indicate that our method is ready to be used for characterizing site-specific
protein glycosylation in complex samples