Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography–tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p

Abstract

Post-translational modification of proteins with glycosylation is of key importance in many biological systems in eukaryotes, influencing fundamental biological processes and regulating protein function. Changes in glycosylation are therefore of interest in understanding these processes and are also useful as clinical biomarkers of disease. The presence of glycosylation can also inhibit protease digestion and lower the quality and confidence of protein identification by mass spectrometry. While deglycosylation can improve the efficiency of subsequent protease digest and increase protein coverage, this step is often excluded from proteomic workflows. Here, we performed a systematic analysis that showed that deglycosylation with peptide-N-glycosidase F (PNGase F) prior to protease digestion with AspN or trypsin improved the quality of identification of the yeast cell wall proteome. The improvement in the confidence of identification of glycoproteins following PNGase F deglycosylation correlated with a higher density of glycosylation

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics

“…Further, in-solution digestion requires low protease-to-substrate ratio (1:50 or lower, m/m) to reduce contamination, thereby operates near-entirely in the enzyme-deficient, diffusion-limited, low-efficiency region (7); slow reaction rates of compromised trypsin in urea amplify such deviation from ideal. In-solution trypsin (2 M urea) digestion has also been extended by combining with other enzymes such as Lys-C (8 M urea) (17), pepsin (18), Asp-N (19), and PNGase F (predigestion (19)). Increasing evidence suggests multispecificity, often via using multiple proteases, is necessary for reliable PSM quantitation of lysates (13,20).…”

“…Extensive PTMs in human signaling TM proteins, such as high-occupancy N-glycosylation, had remained largely a target of observation, but rarely exploited as a means to aid overall peptide formation and detection, partly for fear that PTM enzymes overwhelm real samples (though PNGase F was used to release enriched glycopeptides (77)). Predigestion PNGase F solution incubation indeed identified more proteins in yeast cell wall (19). But emerging complicated N-glycosylation scenarios in mammalian TM proteins suggest, treating peptides post-digestion is more likely efficient and complete than treating proteins (7,66).…”

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

“…Peptides were identified essentially as described using ProteinPilot (AB SCIEX), searching the LudwigNR database (downloaded from http://apcf.edu.au as at 27 January 2012; 16 818 973 sequences; 5 891 363 821 residues) with standard settings: sample type, identification; cysteine alkylation, acrylamide; instrument, TripleTof 5600; species, human, or yeast; ID focus, biological modifications; enzyme, trypsin, or AspN; Search effort, thorough ID. False discovery rate analysis using ProteinPilot was performed on all searches.…”

Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption

“…The Δalg3 deletion strain in SS328 and in the Δire1 strain background displayed a growth phenotype when grown on the SD-URA and SGal-URA plates at 37°C. This effect is most likely unrelated to ERAD or UPR effects, and has been described as being caused by a changed cell wall composition (Bailey and Schulz 2013).…”