Shotgun Glycopeptide Capture Approach Coupled with Mass Spectrometry for Comprehensive Glycoproteomics

Abstract

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatinresistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 ؎ 19 (n ‫؍‬ 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 ؎ 1.6% (n ‫؍‬ 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to

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

“…8 and 9). This glycopeptide selectivity and recovery is comparable with that obtained with hydrazide chemistry capturing tryptic glycopeptides (16). We believe that this IP-NPLC method can be used for isolating glycopeptides at much lower levels because low abundance glycopeptides were effectively recovered from the tryptic digests of periplasmic protein extracts from C. jejuni (Fig.…”

“…Hydrazide chemistry is used to isolate, identify, and quantify N-linked glycopeptides effectively, but this method involves lengthy chemical procedures and does not preserve the glycan moieties thereby losing valuable information on glycan structure and site occupancy (15)(16)(17). Capturing glycopeptides with lectins has been widely used, but restricted specificities and unspecific binding are major drawbacks of this method (18 -21).…”

“…Isolation of glycopeptides from proteolytic digests of complex protein mixtures can greatly enhance the MS signals of glycopeptides using reversed-phase LC-ESI-MS (RPLC-ESI-MS) or MALDI-MS (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Hydrazide chemistry is used to isolate, identify, and quantify N-linked glycopeptides effectively, but this method involves lengthy chemical procedures and does not preserve the glycan moieties thereby losing valuable information on glycan structure and site occupancy (15)(16)(17).…”

N-glycoprotein profiling of lung adenocarcinoma pleural effusions by shotgun proteomics

“…Parallel mass spectrometry of the retained fraction is required to address this issue. 28 Alternatively, trypsin digest before N-GP capture is currently being investigated, but the protocol needs high enzyme amounts to overcome the efficient antiprotease cascades of plasma. Furthermore, N-GP capture of a completely digested protein solution means that one has to fish single N-glycopeptides out of a background of millions of albumin peptides.…”

Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

“…The disadvantages of both these methods are that on the one hand they specifically require the presence of sialylated glycans, and on the other hand they are nonspecific for either N-linked or several of the O-linked types of glycans that can be sialylated. More general glycan oxidative methods have also been introduced (16,17), but despite a longer history, these have so far been demonstrated in practice only for N-glycoproteomics. One alternative is to employ lectin weak affinity chromatography (LWAC) to the proteolytic digest (12,18,19), which can be used to considerable advantage where glycosylation has been simplified to a degree of homogeneity and the lectin is sufficiently specific to exclude unwanted glyco-types (5).…”