Chromatographic properties of deamidated peptides with Asn-Gly sequences in proteomic bottom-up experiments.

Studies surrounding deamidation have relied on the chromatographic and mass spectrometric differentiation of Asn containing peptides and their isomeric Asp and isoAsp products. The development of mass spectrometry analytical techniques and characterization of isomer specific fragmentation patterns has permitted the investigation of some deamidation species but has struggled to remain effective when applied and on complex samples or in high throughput scenarios. On the other hand, chromatographic separations can provide additional information to facilitate detection of deamidation.

Peptide retention time prediction for electrostatic repulsion-hydrophilic interaction chromatography.

Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) is one of the legacy separation tools developed by Dr. Andrew Alpert and has been used for developing unique separation methods of hydrophilic compounds, including peptides. In the past it has been studied using designed peptide libraries to elucidate major features of its separation mechanism, while comprehensive peptide retention modeling for ERLIC is still lacking.

Peptide retention time prediction for hydrophilic interaction liquid chromatography at acidic pH in formic-acid based eluents.

Peptide separation selectivity was evaluated for hydrophilic interaction liquid chromatography (HILIC) ZIC-HILIC, ZIC-cHILIC, and XBridge Amide sorbents using formic acid as eluent additive (pH 2.7). Sequence-specific retention prediction algorithms were trained using retention datasets of ∼30,000 peptides for each column.

Biological Sex Is an Effect Modifier of Allergen-Mediated Alteration of the Lung Proteome.

Asthma exhibits a distinct sex bias in the disease prevalence, severity, and response to therapy. However, sex-related differences in alterations of the lung proteome mediated by aeroallergens critical in asthma, such as house dust mites (HDM), remain unknown. In this study, we define sex-related differences in the lung proteome using an HDM-challenged mouse model by 1D LC-MS/MS.

Paradigm Shift: Major Role of Ion-Pairing-Dependent Size Exclusion Effects in Bottom-Up Proteomics Reversed-Phase Peptide Separations.

Can reversed-phase peptide retention be the same for C8 and C18 columns? or increase for otherwise identical columns with a smaller surface area? Can replacing trifluoroacetic acid (TFA) with formic acid (FA) improve the peak shape? According to our common understanding of peptide chromatography, absolutely not. Surprisingly, a thorough comparison of the peptide separation selectivity of 100 and 120 Å fully porous C18 sorbents to maximize the performance of our in-house proteomics LC-MS/MS setup revealed an unexpectedly higher peptide retentivity for a wider pore packing material, despite it having a smaller surface area. Concurrently, the observed increase in peptide retention─which drives variation in separation selectivity between 100 and 120 Å pore size materials─was more pronounced for smaller peptides.