Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins,
e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for
O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their
O-glycosylation. Here, we report on the site-specific
O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact
O-glycopeptides carrying short mucin-type core-1 and -2
O-glycans, which represent the vast majority of
O-glycans on human blood plasma proteins. Enriched
O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the
O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31
O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel
O-glycosylation sites and regions were identified. In total 23
O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified
O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type
O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma
O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.Human blood plasma harbors arguably the most complex yet also the most informative proteome present in the human body (
1). A significant impact on its clinical relevance and diagnostic potential is attributed to the features and functions of a plethora of proteins (60–80 mg protein per ml plasma), covering a dynamic concentration range of more than ten orders of magnitude (
2). The majority, that is 99%, of these proteins are classical blood plasma proteins, like albumins, (immuno)globulins, clotting factors, and proteins of the complement system; however, also a lower abundant but—no less meaningful—fraction of nonclassical proteins is present that comprises a multitude of cytokines as well as tissue leakage proteins. Several clinical studies could show that qualitative and quantitative alterations of these proteins (and peptides)—analyzed individually or in their entirety as a proteome (or peptidome)—can directly reflect pathophysiological states, and can serve as biomarkers for the onset and progression of a number of diseases (
3–
5). In recent years the focus of in-depth analyses of the human blood plasma proteome has evolved from the identification and quantification of the entire proteome (or peptidome) (
6–
10) toward the analysis of subproteomes like the interactome (
11), phosphoproteome (
12,
13) or the glycoproteome (
14). The latter has received particular interest in recent years, because the majority of blood plasma proteins is
N- and/or
O-glycosylated (
2). Although the comprehensive analysis of the
N-glycoproteome is already quite advanced (
15), even in complex samples like human blood plasma (
16,
17), similar analyses of the
O-glycoproteome - though arguably equally important and relevant - are still lagging behind. The most ubiquitously found and functionally relevant form of
O-glycosylation, as shown by a number of
O-glycan-related (clinical) studies (
18–
23), is the mucin-type
O-glycosyation (
O-GalNAc), in particular the core-1 and core-2 types (
24,
25). The predominantly clustered occurrence of mucin-type
O-glycans on proteins is described to confer overall stability and proteolytic protection (
26). Apart from this global impact, recent studies could link the presence of
O-glycans in the proximity of regulatory domains to proteolysis events involved in protein maturation (proprotein-convertase-processing) (
27). To better understand these protective and regulatory capabilities and to move the mucin-type
O-glycoproteome from form to function comprehensive site-specific
O-glycosylation analyses are required.One of the main obstacles in site-specific mucin-type
O-glycosylation analyses relates to the lack of a predictable
O-glycan consensus-motif within the peptide backbone as it can be found for
N-glycans (
28). The initial attachment of the
N-acetylgalactosamine monosaccharide to the hydroxyl group of either serine or threonine, but also to tyrosine or hydroxylysine, is governed by a family of 20 distinct polypeptide GalNAc-transferase isoenzymes (GalNAc-Ts) with different but partially overlapping peptide specificities and tissue expression patterns. This dynamic regulation, in turn, contributes to the complexity of the mucin-type
O-glycoproteome. However, previous studies could show that mucin-type
O-glycans are primarily attached to serine or threonine in regions with a high content of serine, threonine and proline (Ser/Thr-X-X-Pro, Ser/Thr-P and Pro-Ser/Thr) (
29,
30). As
O-glycosylation is a postfolding event, taking place in the Golgi apparatus, the attachment is depended on protein surface accessibility and is thus predominantly found in coil, turn, and linker regions (
31). Additional confounding factors during mucin-type
O-glycosylation analyses are the clustered occurrence of
O-glycans and the lack of a universal endo-
O-glycosidase that enables the release of intact
O-glycans from the proteins; though, chemical
O-glycan release methods do exist (
28).Mass spectrometry has proven to be the core technique in site-specific
N- and
O-glycosylation analyses. A generic
O-glycoproteomic workflow usually starts with the isolation, enrichment or prefractionation of a single glycoprotein or a group of glycoproteins. In subsequent steps, (glyco)peptides are generated by proteolytic digestion primarily using specific proteases like trypsin. Apart from this, also broad- and nonspecific proteases like Proteinase K or Pronase E were successfully employed in recent years (
32–
34). Essential to nearly every glycoproteomic approach is the removal of high-abundant and interfering nonglycosylated peptides by selective enrichment of the usually lower abundant glycopeptides. The repertoire of glycopeptide enrichment and separation techniques covers different solid phase extraction and chromatography based methods such as hydrophilic liquid interaction chromatography (HILIC) (
35,
36). The most frequently used setup for the measurement of enriched (glyco)peptides is liquid chromatography (LC)
1 coupled online to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Recent advances in instrumentation, in particular the development of electron-transfer/electron-capture dissociation (ETD/ECD) (
37,
38), and high resolution orbital mass analyzers, have paved the way for the mapping of thousands of occupied
N- and
O-glycosylation sites as recently shown (
17,
27). Combined workflows using ETD/ECD fragmentation along with (multistage, MS
n) fragmentation with high- and/or low collisional induced dissociation energy (HCD/CID) can provide compositional (structural) information on the glycan moiety as well as information on the peptide sequence and the glycosylation site (
39,
40). Recent advances in mass spectrometry driven
O-glycoproteomics have been reviewed in detail elsewhere (
41,
42). Owing to the amount and complexity of
O-glycoproteomic data a number of bioinformatic software tools for the prediction of mucin-type
O-glycosylation sites (
27) as well as for the database assisted interpretation and annotation of glycan and glycopeptide fragment spectra have been developed (
43,
44). Moreover, reporting guidelines for collecting, sharing, integrating, and interpreting mass spectrometry based glycomics data have been specified by the MIRAGE consortium (minimum information required for a glycomics experiment) (
45,
46).The aim of our study was to develop a glycoproteomic workflow that allows the explorative nontargeted analysis of
O-glycosylated human blood plasma proteins, which are known to carry mainly short mono- and disialylated mucin-type core-1 and -2
O-glycans. To achieve this, we have combined
O-glycopeptide selective offline-HILIC fractionation of Proteinase K digested peptides with nano-reversed-phase liquid chromatography coupled online to multistage ion-trap mass spectrometry (nanoRP-LC-ESI-IT-MS: CID-MS
2/-MS
3, ETD-MS
2). The workflow has been applied to investigate the mucin-type
O-glycoproteome of a pooled blood plasma sample derived from 20 healthy donors. Based on the mass spectrometric analysis of intact
O-glycopeptides, we were able to characterize the
O-glycosylation (
i.e. peptide, site, and attached
O-glycans) of a number of major human blood glycoproteins, including many acute phase proteins such as fibrinogen and plasminogen. Overall, the site-specific glycosylation analysis of human blood plasma glycopeptides revealed exclusively mono- and disialylated core-1 mucin-type
O-glycopeptides. Interestingly, also a few novel
O-glycosylation sites could be identified.
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