The NJ Mass Spectrometry Discussion Group is pleased to announce our April 25, 2023 Meeting — Online!
NJ MSDG is the second largest mass spectrometry professional association in the nation behind ASMS, with over 1,100 members in the tristate area.
Time:.....7:00 PM | (UTC-05:00) Eastern Time (US & Canada)
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“HILIC/MS – It’s not just for Glycans. Identification of Glycopeptides and Modified Peptides with the Combination of MS and HILIC Retention Modeling”
We have created a HILIC model that can predict the retention of peptides with various modifications. We have shown the ability to chromatographically resolve and quantitate products of deamidation, isomerization, oxidation, O-GlcNAcylation, and glycation. We have derived retention coefficients for these modified amino acids, and have incorporated them into a model that predicts peptide retention on HILIC columns.
The analysis of an extensive range of procainamide tagged N-linked glycans has led to a HILIC retention model for this class of biomolecule. The N-glycan model was combined with the peptide model, described above, to produce a prediction model for glycopeptides. The efficacy of the combined model was evaluated by comparing experimental data obtained via LC-MS analysis of human serum IgGs to those calculated with the model.
We feel that the unified chromatographic model (amino acids, modified amino acids, N-linked glycans) will facilitate the identification and quantification of peptides containing these modifications.
“Unraveling the Released N-Glycan Isomerome Complexity by Implementing High-Resolution Ion Mobility (HRIM) in the HILIC-MS Workflow”
InstantPC-labeled N-linked glycan biantennary standards were analyzed using a combination of flow injection analysis and hydrophilic interaction liquid chromatography (HILIC) separation. Data was acquired on an HRIM MOBIE™ instrument (MOBILion Systems) coupled to a 6545XT QTOF (Agilent Technologies). Accurate mass, isotope spacing, ion mobility arrival time distribution, and Collision Cross Section (CCS) determination were used to identify each N-linked glycan. Each alpha- and beta-linked N-linked glycan isomer structure was verified by analyzing individual standards. Data processing, analysis, relative quantification, and visualization were achieved using HRIM Data Processor, PNNL Preprocessor, and Protein Metrics Byos® Software.
First, flow injection analysis of 28 InstantPC-labeled N-linked glycan compositions (of which, 12 have isomeric linkages) was conducted to determine the HRIM-MS profiles of each N-glycan. For each labeled N-linked glycan, accurate mass, isotope spacing, ion mobility arrival time distribution, and Collision Cross Section (CCS) values were determined with a minimum of triplicate measurements. N-linked glycan detection, identity validation, and relative quantitation were conducted, generating an N-linked glycan feature library used in later HILIC-HRIM-MS analytical workflows. We demonstrate that HRIM-MS allows for the separation, confident identification, and quantification of all 28 N-linked glycan species. Further, all 12 N-linked glycan isomers could be resolved into multiple ion mobility peaks in the gas phase, without any LC separation. Rapid N-linked glycan profiling was further demonstrated by analyzing a human IgG-released InstantPC-labeled N-linked glycan library. Altogether, our flow injection results demonstrate that HRIM-MS offers excellent potential for rapidly and confidently revealing complex glycoform profiles.
Second, we implemented HRIM in the traditional HILIC-MS analytical workflow for released N-linked glycans, intending to test the complementarity of HILIC and HRIM separation. Varying HILIC run times (60-, and 15-minute gradients) were tested to assess the ability of HRIM to resolve N-linked glycans in the gas phase while HILIC separation times were gradually reduced. All three HILIC gradients resulted in comparable relative N-linked glycan species coverage and isomer separation, with consistent and reproducible relative quantitation based on extracted ion mobiligrams. Our results demonstrate that HRIM-MS provides high-quality and rapid N-linked glycan feature identification (including isomeric separation) and relative quantitation. Leveraging accurate and reproducible CCS determination, adding HRIM to a traditional LC-MS method provides a solution for confidently fingerprinting glycosylation profiles in biotherapeutics, enabling a greater depth in N-linked glycan feature detection or increased analytical throughput.
Three bullet points about the talk:
- Traditional mAb-released N-glycan analysis results in a 15-60% ambiguity rate based on the inability to resolve isomers and reporting of compositions only.
- HRIM-MS allows for the separation, confident identification, and quantification of released N-glycan species, including isomer resolution, without any LC separation.
- Leveraging accurate and reproducible CCS determination, HRIM addition to a HILIC-MS workflow provides confident fingerprinting of glycosylation profiles in biotherapeutics while enabling increased analytical throughput.
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