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Abstract Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid‐phase separations. Native proteomics should provide the most accurate bird's‐eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well‐purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra‐high mass range (UHMR) Orbitrap mass spectrometer. The nCZE‐MS technique enabled the measurement of a 115‐kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE‐MS analysis of anE.colicell lysate detected 72 proteoforms or protein complexes in a mass range of 30–400 kDa in a single run while consuming only 50‐ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes. 

Abstract Mass spectrometry (MS)‐based top‐down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post‐translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high‐resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS‐PAGE‐based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI‐MS) with capillary zone electrophoresis (CZE)‐MS/MS for high‐resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS‐PAGE gel and follow‐up cleanup as well as CZE‐MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high‐resolution separation and characterization of histone proteoforms. SDS‐PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high‐resolution separations of SDS‐PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems. 

Abstract We present a large‐scale top‐down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size‐exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post‐translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post‐translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N‐ and C‐terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub‐cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub‐cellular localization signal predictions. This large‐scale analysis illustrates the power of top‐down proteoform identification of post‐translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins. 

The first example of top-down proteomics of nanoparticle protein coronas using capillary isoelectric focusing-mass spectrometry. 

Top-down proteomics of colorectal cancer cells provides proteoform-level knowledge about cancer metastasis. 

Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) identify proteoforms without pretreatment of enzyme proteolysis. A universal sample preparation method that can efficiently extract protein, reduce sample loss, maintain protein solubility, and be compatible with following up liquid-phase separation, MS, and tandem MS (MS/MS) is vital for large-scale proteoform characterization. Membrane ultrafiltration (MU) was employed here for buffer exchange to efficiently remove the sodium dodecyl sulfate (SDS) detergent in protein samples used for protein extraction and solubilization, followed by capillary zone electrophoresis (CZE)-MS/MS analysis. The MU method showed good protein recovery, minimum protein bias, and nice compatibility with CZE-MS/MS. Single-shot CZE-MS/MS analysis of an Escherichia coli sample prepared by the MU method identified over 800 proteoforms.