Coupling High-Field Asymmetric Ion Mobility Spectrometry with Capillary Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry Improves Protein Identifications in Bottom-Up Proteomic Analysis of Low Nanogram Samples

Abstract

In this work, we pioneered the assessment of coupling high-field asymmetric waveform ion mobility spectrometry (FAIMS) with ultrasensitive capillary electrophoresis hyphenated with tandem mass spectrometry (CE-MS/MS) to achieve deeper proteome coverage of low nanogram amounts of digested cell lysates. An internal stepping strategy using three or four compensation voltages per analytical run with varied cycle times was tested to determine optimal FAIMS settings and MS parameters for the CE-FAIMS-MS/MS method. The optimized method applied to bottom-up proteomic analysis of 1 ng of HeLa protein digest standard identified 1314 ± 30 proteins, 4829 ± 200 peptide groups, and 7577 ± 163 peptide spectrum matches (PSMs) corresponding to a 16, 25, and 22% increase, respectively, over CE-MS/MS alone, without FAIMS. Furthermore, the percentage of acquired MS/MS spectra that resulted in PSMs increased nearly 2-fold with CE-FAIMS-MS/MS. Label-free quantitation of proteins and peptides was also assessed to determine the precision of replicate analyses from FAIMS methods with increased cycle times. Our results also identified from 1 ng of HeLa protein digest without any prior enrichment 76 ± 9 phosphopeptides, 18% of which were multiphosphorylated. These results represent a 46% increase in phosphopeptide identifications over the control experiments without FAIMS yielding 2.5-fold more multiphosphorylated peptides.

HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics

“…In the hyperSCP workflow, high labeling efficiency is necessary for accurate quantification. To determine the SILAC labeling efficiency, the cell samples were prepared in bulk, 38 analyzed by nanoLC−MS, and databasesearched using PD 2.5, with the label 13 C 6 15 N 2 (+8.014 Da, K) and label 13 C 6 15 N 4 (+10.008 Da, R) selected as dynamic modifications. The results indicated that 97% of lysines and 98% of arginines were labeled in the SILAC-heavy cells.…”

“…HeLa, A549, K562, and HFL1 cells were obtained from ATCC (Manassas, VA) and cultured at 37 °C with 5% CO 2 using cell culture medium with 10% fetal bovine serum and 1% penicillin/streptomycin. Non-SILAC ("light") cells were cultured in regular RPMI 1640 medium, while the SILAC-labeled ("heavy") cells were cultured in the RPMI 1640 medium in which the lysine and arginine were replaced with 13 C 6 15 N 2 L-lysine-2HCl and 13 C 6 15 N 4 L-arginine-HCl. The cells were cultured as reported 38 and split upon reaching ∼70% confluency.…”

“…Liquid chromatography–mass spectrometry (LC–MS) is increasingly utilized for unbiased analysis of the proteome of individual cells . The increased speed and sensitivity of modern mass spectrometers, , along with improved sample preparation, − separations, − and experimental design − have allowed for deeper analysis in single-cell proteomics (SCP). SCP can be broadly categorized as advancing along two tracks: label-free and label-based workflows.…”

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (mid‐2018–2022)

“…Combination of sheathless CE‐MS/MS with the high‐field asymmetric waveform ion mobility spectrometry that functions as an ion‐selective device, reduced background noise, and increased S/N of the CE‐MS analyses [118]. The optimized method with high‐field asymmetric waveform ion mobility spectrometry identified from 1 ng of standard HeLa protein digest 16%, 25%, and 22% of more proteins, peptide groups, and peptide spectrum matches, respectively, than solely CE‐MS/MS.…”

High-Field Asymmetric Waveform Ion Mobility Spectrometry: Practical Alternative for Cardiac Proteome Sample Processing

“…Combining multiple compensation voltages (CVs) within a single run or between runs improves whole proteome coverage in cell lysates. , For example, Pfammatter et al observed a 30% gain in unique peptide identification using FAIMS compared with non-FAIMS analysis of HEK 293 cells . Other studies using HeLa protein digest such as Johnson KR et al, Greguš M et al, and Cong Y et al concurrently showed the addition of FAIMS had significantly increased protein and peptide identifications than without FAIMS. − In a few recent studies working with various tissue samples, including brain autopsies, tumor biopsies, and paraffin embedded tissues (lymph node, lung, and prostate), the addition of FAIMS also showed an enhanced detection of nonredundant proteoforms, increased proteome sensitivity, or substantially reduced sample handling. As the cardiac proteome consists of large dynamic range, it remains unclear whether FAIMS can detect peptides and the less abundant proteins from the cardiac proteome that is comparable with previous established fractionation methods.…”