Journal Article

Multidimensional separation and analysis of alpha-1-acid glycoprotein N-glycopeptides using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and nano-liquid chromatography tandem mass spectrometry

Chandler, Kevin Brown, Marrero Roche, Daniel E., Sackstein, Robert

2023

Overview

Bottom-up nLC-MS/MS-based glycoprotein mass spectrometry workflows rely on the generation of a mixture of non-glycosylated and glycosylated peptides via proteolysis of glycoproteins. Such methods are challenged by suppression of hydrophilic glycopeptide ions by more abundant, hydrophobic, and readily ionizable non-glycosylated peptides. Commercially available high-field asymmetric waveform ion mobility spectrometry (FAIMS) devices have recently been introduced and present a potential benefit for glycoproteomic workflows by enabling orthogonal separation of non-glycosylated peptides and glycopeptides following chromatographic separation, and prior to MS/MS analysis. However, knowledge is lacking regarding optimal FAIMS conditions for glycopeptide analyses. Here, we document optimal FAIMS compensation voltages for the transmission and analysis of human alpha-1-acid glycoprotein (AGP) tryptic N -glycopeptide ions. Further, we evaluate the effect of FAIMS on AGP glycopeptide assignment confidence by comparing the number of assigned glycopeptides at different confidence levels using a standard nLC-MS/MS method or an otherwise identical method employing FAIMS. Optimized methods will potentiate glycoproteomic analyses by increasing the number of unique glycopeptide identifications and the confidence of glycopeptide assignments. Data are available via ProteomeXchange with identifier PXD036667. Graphical Abstract Analysis of alpha-1-acid glycoprotein (AGP) tryptic digests via nLC-FAIMS-MS/MS ( top ) led to the establishment of ideal FAIMS voltages for the analysis of AGP N -glycopeptides ( bottom ), suggesting that FAIMS can improve the depth of glycoproteome characterization. Pairs of CV magnitudes are shown along the x -axis

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: ABC Highlights: authored by Rising Stars and Top Experts / Analytical Chemistry / Antibiotics / Apoptosis Regulatory Proteins / Asymmetry / Biochemistry / Characterization and Evaluation of Materials / Chemistry / Chemistry and Materials Science / chromatography / Confidence intervals / Food Science / Glycopeptides / Glycoproteins / glycoproteomics / glycosylation / Humans / hydrophilicity / Hydrophobicity / Ion Mobility Spectrometry / Ionic mobility / Ions / Ions - chemistry / Laboratory Medicine / Liquid chromatography / Mass spectrometry / Mass spectroscopy / Mobility / Monitoring/Environmental Analysis / Orosomucoid / Paper in Forefront / Peptides / Peptides - chemistry / Physiological aspects / Proteolysis / Scientific imaging / Separation / Spectroscopy / tandem mass spectrometry / Tandem Mass Spectrometry - methods / Waveforms