Coupling High-Field Asymmetric Waveform Ion Mobility Spectrometry with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has emerged as an essential technique for top-down proteomics (TDP), providing superior separation efficiency and high detection sensitivity for proteoform analysis. Here, we aimed to further enhance the performance of CZE-MS/MS for TDP via coupling online gas-phase proteoform fractionation using high-field asymmetric waveform ion mobility spectrometry (FAIMS). When the compensation voltage (CV) of FAIMS was changed from −50 to 30 V, the median mass of identified proteoforms increased from less than 10 kDa to about 30 kDa, suggesting that FAIMS can efficiently fractionate proteoforms by their size. CZE-FAIMS-MS/MS boosted the number of proteoform identifications from a yeast sample by nearly 3-fold relative to CZE-MS/MS alone. It particularly benefited the identification of relatively large proteoforms, improving the number of proteoforms in a mass range of 20–45 kDa by 6-fold compared to CZE-MS/MS alone. FAIMS fractionation gained nearly 20-fold better signal-to-noise ratios of randomly selected proteoforms than no FAIMS. We expect that CZE-FAIMS-MS/MS will be a useful tool for further advancing the sensitivity and coverage of TDP. This work shows the first example of coupling CE with ion mobility spectrometry (IMS) for TDP

Coupling High-Field Asymmetric Waveform Ion Mobility Spectrometry with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has emerged as an essential technique for top-down proteomics (TDP), providing superior separation efficiency and high detection sensitivity for proteoform analysis. Here, we aimed to further enhance the performance of CZE-MS/MS for TDP via coupling online gas-phase proteoform fractionation using high-field asymmetric waveform ion mobility spectrometry (FAIMS). When the compensation voltage (CV) of FAIMS was changed from −50 to 30 V, the median mass of identified proteoforms increased from less than 10 kDa to about 30 kDa, suggesting that FAIMS can efficiently fractionate proteoforms by their size. CZE-FAIMS-MS/MS boosted the number of proteoform identifications from a yeast sample by nearly 3-fold relative to CZE-MS/MS alone. It particularly benefited the identification of relatively large proteoforms, improving the number of proteoforms in a mass range of 20–45 kDa by 6-fold compared to CZE-MS/MS alone. FAIMS fractionation gained nearly 20-fold better signal-to-noise ratios of randomly selected proteoforms than no FAIMS. We expect that CZE-FAIMS-MS/MS will be a useful tool for further advancing the sensitivity and coverage of TDP. This work shows the first example of coupling CE with ion mobility spectrometry (IMS) for TDP

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of Mouse Brain Integral Membrane Proteins

Mass spectrometry (MS)-based top-down characterization of integral membrane proteins (IMPs) is crucial for understanding their functions in biological processes. However, it is technically challenging due to their low solubility in typical MS-compatible buffers. In this work, for the first time, we developed an efficient capillary zone electrophoresis (CZE)-tandem MS (MS/MS) method for the top-down proteomics (TDP) of IMPs enriched from mouse brains. Our technique employs a sample buffer containing 30% (v/v) formic acid and 60% (v/v) methanol for solubilizing IMPs and utilizes a separation buffer of 30% (v/v) acetic acid and 30% (v/v) methanol for maintaining the solubility of IMPs during CZE separation. Single-shot CZE-MS/MS identified 51 IMP proteoforms from the mouse brain sample. Coupling size exclusion chromatography (SEC) to CZE-MS/MS enabled the identification of 276 IMP proteoforms from the mouse brain sample containing 1–4 transmembrane domains. This proof-of-concept work demonstrates the high potential of CZE-MS/MS for the large-scale TDP of IMPs

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of Mouse Brain Integral Membrane Proteins

Mass spectrometry (MS)-based top-down characterization of integral membrane proteins (IMPs) is crucial for understanding their functions in biological processes. However, it is technically challenging due to their low solubility in typical MS-compatible buffers. In this work, for the first time, we developed an efficient capillary zone electrophoresis (CZE)-tandem MS (MS/MS) method for the top-down proteomics (TDP) of IMPs enriched from mouse brains. Our technique employs a sample buffer containing 30% (v/v) formic acid and 60% (v/v) methanol for solubilizing IMPs and utilizes a separation buffer of 30% (v/v) acetic acid and 30% (v/v) methanol for maintaining the solubility of IMPs during CZE separation. Single-shot CZE-MS/MS identified 51 IMP proteoforms from the mouse brain sample. Coupling size exclusion chromatography (SEC) to CZE-MS/MS enabled the identification of 276 IMP proteoforms from the mouse brain sample containing 1–4 transmembrane domains. This proof-of-concept work demonstrates the high potential of CZE-MS/MS for the large-scale TDP of IMPs

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample

Mass spectrometry (MS)-based top-down proteomics (TDP) has revolutionized biological research by measuring intact proteoforms in cells, tissues, and biofluids. Capillary zone electrophoresis–tandem MS (CZE-MS/MS) is a valuable technique for TDP, offering a high peak capacity and sensitivity for proteoform separation and detection. However, the long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which is a crucial aspect for large-scale studies. This work investigated the long-term qualitative and quantitative reproducibility of CZE-MS/MS for TDP for the first time, focusing on a yeast cell lysate. Over 1000 proteoforms were identified per run across 62 runs using one linear polyacrylamide (LPA)-coated separation capillary, highlighting the robustness of the CZE-MS/MS technique. However, substantial decreases in proteoform intensity and identification were observed after some initial runs due to proteoform adsorption onto the capillary inner wall. To address this issue, we developed an efficient capillary cleanup procedure using diluted ammonium hydroxide, achieving high qualitative and quantitative reproducibility for the yeast sample across at least 23 runs. The data underscore the capability of CZE-MS/MS for large-scale quantitative TDP of complex samples, signaling its readiness for deployment in broad biological applications. The MS RAW files were deposited in ProteomeXchange Consortium with the data set identifier of PXD046651

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample

Mass spectrometry (MS)-based top-down proteomics (TDP) has revolutionized biological research by measuring intact proteoforms in cells, tissues, and biofluids. Capillary zone electrophoresis–tandem MS (CZE-MS/MS) is a valuable technique for TDP, offering a high peak capacity and sensitivity for proteoform separation and detection. However, the long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which is a crucial aspect for large-scale studies. This work investigated the long-term qualitative and quantitative reproducibility of CZE-MS/MS for TDP for the first time, focusing on a yeast cell lysate. Over 1000 proteoforms were identified per run across 62 runs using one linear polyacrylamide (LPA)-coated separation capillary, highlighting the robustness of the CZE-MS/MS technique. However, substantial decreases in proteoform intensity and identification were observed after some initial runs due to proteoform adsorption onto the capillary inner wall. To address this issue, we developed an efficient capillary cleanup procedure using diluted ammonium hydroxide, achieving high qualitative and quantitative reproducibility for the yeast sample across at least 23 runs. The data underscore the capability of CZE-MS/MS for large-scale quantitative TDP of complex samples, signaling its readiness for deployment in broad biological applications. The MS RAW files were deposited in ProteomeXchange Consortium with the data set identifier of PXD046651

Predicting Electrophoretic Mobility of Proteoforms for Large-Scale Top-Down Proteomics

Large-scale top-down proteomics characterizes proteoforms in cells globally with high confidence and high throughput using reversed-phase liquid chromatography (RPLC)–tandem mass spectrometry (MS/MS) or capillary zone electrophoresis (CZE)–MS/MS. The false discovery rate (FDR) from the target–decoy database search is typically deployed to filter identified proteoforms to ensure high-confidence identifications (IDs). It has been demonstrated that the FDRs in top-down proteomics can be drastically underestimated. An alternative approach to the FDR can be useful for further evaluating the confidence of proteoform IDs after the database search. We argue that predicting retention/migration time of proteoforms from the RPLC/CZE separation accurately and comparing their predicted and experimental separation time could be a useful and practical approach. Based on our knowledge, there is still no report in the literature about predicting separation time of proteoforms using large top-down proteomics data sets. In this pilot study, for the first time, we evaluated various semiempirical models for predicting proteoforms’ electrophoretic mobility (μef) using large-scale top-down proteomics data sets from CZE–MS/MS. We achieved a linear correlation between experimental and predicted μef of E. coli proteoforms (R2 = 0.98) with a simple semiempirical model, which utilizes the number of charges and molecular mass of each proteoform as the parameters. Our modeling data suggest that the complete unfolding of proteoforms during CZE separation benefits the prediction of their μef. Our results also indicate that N-terminal acetylation and phosphorylation both decrease the proteoforms’ charge by roughly one charge unit