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We report femtosecond time-resolved measurements of the McLa!erty rearrangement following the strong-field tunnel ionization of 2-pentanone, 4-methyl-2-pentanone, and 4,4-dimethyl-2-pentanone. The pump−probe-dependent yields of the McLa!erty product ion are fit to a biexponential function with fast ("100 fs) and slow ("10 ps) time constants, the latter of which is faster for the latter two compounds. Following nearly instantaneous ionization, the fast time scale is associated with rotation of the molecule to a six-membered cyclic intermediate that facilitates transfer of the !-hydrogen, while the "50−100 times longer time scale is associated with a "-bond rearrangement and bond cleavage between the #- and $-carbons to produce the enol cation. These experimental measurements are supported by ab initio molecular dynamics trajectories, which further confirm the time scale of this important stepwise reaction in mass spectrometry. 

Characterization of histone proteoforms with various post‐translational modifications (PTMs) is critical for a better understanding of functions of histone proteoforms in epigenetic control of gene expression. Mass spectrometry (MS)‐based top‐down proteomics (TDP) is a valuable approach for delineating histone proteoforms because it can provide us with a bird's‐eye view of histone proteoforms carrying diverse combinations of PTMs. Here, we present the first example of coupling capillary zone electrophoresis (CZE), ion mobility spectrometry (IMS), and MS for online multi‐dimensional separations of histone proteoforms. Our CZE‐high‐field asymmetric waveform IMS (FAIMS)‐MS/MS platform identified 366 (ProSight PD) and 602 (TopPIC) histone proteoforms from a commercial calf histone sample using a low microgram amount of histone sample as the starting material. CZE‐FAIMS‐MS/MS improved the number of histone proteoform identifications by about 3 folds compared to CZE‐MS/MS alone (without FAIMS). The results indicate that CZE‐FAIMS‐MS/MS could be a useful tool for comprehensive characterization of histone proteoforms with high sensitivity.

 

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

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. 

Mass spectrometry (MS)-based top-down proteomics (TDP) requires high-resolution separation of proteoforms before electrospray ionization (ESI)-MS and tandem mass spectrometry (MS/MS). Capillary isoelectric focusing (cIEF)-ESI-MS and MS/MS could be an ideal method for TDP because cIEF can enable separation of proteoforms based on their isoelectric points (pIs) with ultra-high resolution. cIEF-ESI-MS has been well-recognized for protein characterization since 1990s. However, the widespread adoption of cIEF-MS for the characterization of proteoforms had been impeded by several technical challenges, including the lack of highly sensitive and robust ESI interface for coupling cIEF to MS, ESI suppression of analytes from ampholytes, and the requirement of manual operations. In this mini review, we summarize the technical improvements of cIEF-ESI-MS for characterizing proteoforms and highlight some recent applications to hydrophobic proteins, urinary albumin variants, charge variants of monoclonal antibodies, and large-scale TDP of complex proteomes. 

Mass spectrometry (MS)-based proteomics has enabled the identification and quantification of thousands of proteins from complex proteomes in a single experiment. However, its performance for mass-limited proteome samples ( e.g. , single cells and tissue samples from laser capture microdissection) is still not satisfying. The development of novel proteomic methodologies with better overall sensitivity is vital. During the last several years, substantial technical progress has been achieved for the preparation and liquid-phase separation-MS characterization of mass-limited proteome samples. In this review, we summarize recent technological progress of sample preparation, liquid chromatography (LC)-MS, capillary zone electrophoresis (CZE)-MS and MS instrumentation for bottom-up proteomics of trace biological samples, highlight some exciting applications of the novel techniques for single-cell proteomics, and provide a very brief perspective about the field at the end.