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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

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Abstract

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

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [26] partner repository with the dataset identifier PXD036667.

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Acknowledgements

DEMR was supported by the QBIC program at Florida International University. KBC is supported by NIH NHLBI K12 (5K12HL141953-03). RS is supported by NCI U01 CA225730 (Alliance of Glycobiologists for Cancer Research).

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Authors and Affiliations

  1. Translational Glycobiology Institute, Department of Translational Medicine, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, 33199, USA

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

  2. Biomolecular Sciences Institute, Florida International University, 11200 SW 8th St., Miami, FL, 33199, USA

    Kevin Brown Chandler

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Correspondence to Kevin Brown Chandler.

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Supplementary Information

Below is the link to the electronic supplementary material.

216_2022_4435_MOESM1_ESM.xlsx

Electronic Supplementary Material Figure S1: FAIMS Analyses of Unenriched, Tryptic AGP digests Over a Range of Compensation Voltages. (XLSX 62 KB)

216_2022_4435_MOESM2_ESM.xlsx

Electronic Supplementary Material Figure S2: Comparison of CV Combinations in Unenriched AGP (Unique Glycopeptides). (XLSX 128 KB)

216_2022_4435_MOESM3_ESM.xlsx

Electronic Supplementary Material Figure S3: Comparison of a Standard nLC-MS/MS Method or an Identical Method with FAIMS, Alternating Continuously between Three Compensation Voltages (CV) (-35V/ -40V/ -50V). (XLSX 4209 KB)

216_2022_4435_MOESM4_ESM.pdf

Electronic Supplementary Material Figure S4: Venn diagrams comparing (a) of unique (and shared, overlapping) N-glycopeptides between -35V, -40V, and -50V FAIMS settings. (b) unique (and shared, overlapping) N-glycopeptides between -35V and -50V FAIMS settings, by charge state (z = 2+ to 5+). (PDF 300 KB)

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Chandler, K.B., Marrero Roche, D.E. & Sackstein, R. 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. Anal Bioanal Chem 415, 379–390 (2023). https://doi.org/10.1007/s00216-022-04435-3

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  • DOI: https://doi.org/10.1007/s00216-022-04435-3

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