Enhanced Separation of Intact Proteins and Proteoforms by CZE-MS Using Sulfobetaine-Modified Poly(α-L-lysine)-Based Multilayer Coatings for EOF Adjustment
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)
SUBMITTER: 
Alisa Höchsmann
LAB HEAD: Christian Neusüß
PROVIDER: PXD061793 | Pride | 2025-10-19
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| PSPSL-08_YEAST_Run1.pdResult | Other | |||
| PSPSL-08_YEAST_Run1.raw | Raw | |||
| PSPSL-08_YEAST_Run2.pdResult | Other | |||
| PSPSL-08_YEAST_Run2.raw | Raw | |||
| PSPSL-08_YEAST_Run3.pdResult | Other |
Items per page: 5 1 - 5 of 74 |
Similar Datasets
Project description:Analysis of human Caucasian colon adenocarcinoma cells (CaCo-2) using a novel selective comprehensive online nanoLCxCZE-MS platform to increase the number of detected proteoforms. The dataset also contains 1D nanoLC-MS and 1D CZE-MS reference measurements.
2025-10-20 | PXD064728 | Pride
Project description:In order to elucidate the influence of in-source ion activation on artificial proteoform truncation, intact proteoforms from Escherichia coli lysate (enriched by proteoforms smaller than 20 kDa) were analyzed using LC-FAIMS-MS/MS with varying in-source ion activation energies.
2025-11-11 | PXD066719 | Pride
Project description:Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has become a valuable analytical technique in top-down proteomics (TDP) in recent years. CZE-MS/MS-based TDP typically employs separation capillaries with neutral coatings (i.e., linear polyacrylamide, LPA). However, issues related to separation resolution and reproducibility remain with the LPA-coated capillaries due to the unavoidable non-specific protein adsorption onto the capillary wall. Cationic coatings can be critical alternatives to LPA coating for CZE-MS/MS-based TDP due to the electrostatic repulsion between the positively charged capillary inner wall and proteoform molecules in the acidic separation buffer. Unfortunately, there are only very few studies using cationic coating-based CZE-MS/MS for TDP studies. In this work, we aimed to develop a simple and efficient approach for preparing separation capillaries with cationic coating, i.e., poly(acrylamide-co-(3-acrylamidopropyl)trimethylammonium chloride, PAMAPTAC for CZE-MS/MS-based TDP. The PAMAPTAC coating-based CZE-MS produced significantly better separation resolution of proteoforms compared to traditionally used LPA-coated approach. It achieved reproducible separation and measurement of simple proteoform mixture and a complex proteome sample (i.e., a yeast cell lysate) regarding migration time, proteoform intensity, and the number of proteoform identifications. The PAMAPTAC coating-based CZE-MS enabled the detection of large proteoforms (≥30 kDa) from the yeast cell lysate reproducibly without any size-based prefractionation. Interestingly, the electrophoretic mobility of proteoforms using the PAMAPTAC coating can be predicted accurately using a simple semiempirical model. The results render the PAMAPTAC coating as a valuable alternative to the LPA coating to advance CZE-MS-based TDP towards high-resolution separation and highly reproducible measurement of proteoforms in complex samples.
2024-10-15 | PXD051862 | Pride
Project description:Top-down proteomics (TDP) has made significant advances in the past, and a plethora of sample preparation workflows have been developed. Here, we systematically investigated the influence of different sample preparation steps on proteoform and protein identifications, including cell lysis, reduction and alkylation, proteoform enrichment, purification, and fractionation. We found that all steps in sample preparation influence the subset of proteoforms identified, e.g., their number, confidence, physicochemical properties, and artificially generated modifications. The various sample preparations resulted in complementary identifications, significantly increasing the depth of the analysis. Overall, 15,089 proteoforms from 2,780 protein accessions of human Caco-2 cells were identified, providing the most comprehensive dataset of this cell line up to now. The results from our study can serve as a guideline for designing and adapting TDP sample preparation strategies to particular research questions.
2024-08-08 | PXD049969 | Pride
Project description:Middle-down proteomics is an analytical approach whereby protein samples are digested with proteases such as Glu-C to generate large peptides (>3 kDa) that are then analyzed by mass spectrometry. This method is useful for characterizing high-molecular-weight proteins that are difficult to detect by top-down proteomics, in which intact proteins are analyzed by mass spectrometry. In this study, we applied GeLC-FAIMS-MS, a multidimensional separation workflow that combines gel-based prefractionation with LC-FAIMS Orbitrap mass spectrometry, for deep middle-down proteomics. Middle-down peptides obtained under the condition of optimized limited Glu-C digestion were first size-fractionated by polyacrylamide gel electrophoresis, followed by C4 reversed-phase liquid chromatography separation and additional ion mobility fractionation, resulting in a significant increase in peptide length detectable by mass spectrometry. In addition to global analysis, the GeLC-FAIMS concept can also be applied to targeted middle-down proteomics, where only proteins in the desired molecular weight range are gel-fractionated and their Glu-C digestion products are analyzed, as demonstrated by targeted analysis of integrins in exosomes. In-depth middle-down proteomics achieved by global and targeted GeLC-FAIMS-MS allows the exploration of proteoform information not covered by conventional top-down proteomics via increase in the number of detectable proteoforms and improvement in sequence coverage.
2024-11-20 | PXD041358 | Pride
Project description:In top-down (TD) proteomics, fractionation prior to mass spectrometric (MS) analysis is a crucial step for the high confidence identification of proteoforms and increased sample depth. In addition to liquid phase separations, gas-phase fractionation strategies such as field asymmetric ion mobility spectrometry (FAIMS) have been shown to be highly beneficial in TD proteomics. However, the need for multiple injections using different compensation voltages (CV) leads to a huge increase in measurement time and the amount of sample required. Therefore, we here investigated for the first time the use of internal CV stepping for single shot TD analysis, i.e., the application of multiple CVs per acquisition. In addition, MS parameters were optimized for the individual CVs since different CVs target certain mass ranges. For example, small proteoforms identified mainly with lower CVs can be identified with a lower resolution and number of microscans than larger proteins identified mainly with higher CVs. We investigated the optimal combination and number of CVs for different gradient lengths and validated the optimized settings with the low molecular weight proteome of CaCo 2 cells obtained by different sample preparation techniques. Compared to measurements without FAIMS, both the number of identified protein groups (+60-94%) and proteoforms (+46-127%), and their confidence were significantly increased, while the measurement time remained identical. In total, we identified 684 protein groups and 2,675 proteoforms from CaCo-2 cells in less than 24 hours using the optimized multi-CV method.
2022-02-28 | PXD029792 | Pride
Project description:Mass spectrometry (MS)-based spatially resolved top-down proteomics (TDP) of tissues is crucial for understanding the roles played by microenvironmental heterogeneity in the biological functions of organs and for discovering new proteoform biomarkers of diseases. There are few published spatially resolved TDP studies. One of the challenges relates to the limited performance of TDP for the analysis of spatially isolated samples using, for example, laser capture microdissection (LCM) because those samples are usually mass-limited. We present the first pilot study of LCM-capillary zone electrophoresis (CZE)-MS/MS for spatially resolved TDP and used zebrafish brain as the sample. The LCM-CZE-MS/MS platform employed a non-ionic detergent and a freeze-thaw method for efficient proteoform extraction from LCM isolated brain sections followed by CZE-MS/MS without any sample cleanup step, ensuring high sensitivity. Over 400 proteoforms were identified in a CZE-MS/MS analysis of one LCM brain section via consuming the protein content of roughly 250 cells. We observed drastic differences in proteoform profiles between two LCM brain sections isolated from the optic tectum (Teo) and telencephalon (Tel) regions. Proteoforms of three proteins (npy, penkb, and pyya) having neuropeptide hormone activity were exclusively identified in the isolated Tel section. Proteoforms of reticulon, myosin, and troponin were almost exclusively identified in the isolated Teo section, and those proteins play essential roles in visual and motor activities. The proteoform profiles accurately reflected the main biological functions of the Teo and Tel regions of the brain. Additionally, hundreds of post-translationally modified proteoforms were identified.
2023-03-11 | PXD027741 | Pride
Project description:We developed a novel sample preparation method by combining Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (PEPPI-MS) fractionation with Anion-Exchange disc-assisted Sequential sample Preparation (AnExSP) purification, and evaluated its performance in TDP analysis by comparing it with a conventional PEPPI workflow using Methanol-Chloroform-Water (MCW) precipitation.
2022-09-26 | PXD034353 | Pride
Project description: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 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 datasets. In this pilot study, for the first time, we evaluated various semi-empirical models for predicting proteoforms’ electrophoretic mobility (µef) using large-scale top-down proteomics datasets from CZE-MS/MS. We achieved a linear correlation between experimental and predicted µef of E. coli proteoforms (R2=0.98) with a simple semi-empirical 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 proteoforms’ charge by roughly one charge unit.
2020-02-17 | PXD017265 | Pride
Project description:Here, we describe the development of a labeling strategy for TDP targeting thiol groups of cysteine residues with iodoTMTsixplex. While this method inherently excludes the quantification of cysteine-free proteoforms, it provides the opportunity of sixplexing and the implementation of multidimensional separation schemes. The approach was tested using both a high/low-pH RP-LC and a gel-eluted liquid fraction entrapment electrophoresis (GelFrEE) x RP-LC separation. Over- and underlabeling rates were determined and MS/MS parameters were optimized to enable parallel protein identifications and quantification. Finally, a complex two proteome interference model was applied to demonstrate the high accuracy of the developed quantification method.
2021-08-06 | PXD026310 | Pride