Authors: Yun Yang | Ruijun Tian
Source: PubMed (review, 2020-10-08)
Field: Chemistry — Spectroscopy

[Recent progress in capillary electrophoresis-based high-sensitivity proteomics]. In recent years, proteomic techniques have undergone rapid progress in terms of sample pretreatment, separation, and mass spectrometry (MS) detection. The current MS-based proteomic techniques can be used to identify up to 10000 proteins both qualitatively and quantitatively within a few hours. However, the current mainstream proteomic approaches do not fulfill the need to analyze minute amounts of biological samples, especially rare cells and single mammalian cells. Capillary electrophoresis (CE)-based separation offers many advantages, such as narrow peaks, high separation efficiency, and low sample requirement, which make it an ideal separation approach for combination with high-resolution MS. We have reviewed the state-of-the-art development of integrated and online sample preparation methods and nanoscale liquid chromatography-mass spectrometry (nanoLC-MS) for high-sensitivity proteomics, and described the associated challenges. Integrated and online sample preparation methods can minimize sample loss and improve lysis and digestion efficiencies. The simple and integrated spintip-based proteomics technology (SISPROT) developed by our group has shown robust performance for the comprehensive profiling of various types of samples and the sensitive analysis of small numbers of cells, down to a few hundred. A few groups have applied integrated/online sample preparation methods, such as nanodroplet processing in one pot for trace samples (nanoPOTS) and integrated proteome analysis system for one cell (iPAD-1), to achieve the identification of hundreds of proteins from a single HeLa cell. We propose that one of the key technical challenges in this field is that the performance of current nanoLC separation techniques cannot match modern high-resolution MS techniques, with ultrahigh scan rates of over 40 Hz; therefore, the insufficient chromatographic performance results in reduced utilization of MS/MS scan capacity. Wide chromatographic peaks result in insufficient precursors to trigger MS/MS scans and redundant sampling, irrespective of whether dynamic exclusion has been enabled. In view of the above mentioned technical challenges, we have focused on discussing the unique technical advantages and potential opportunities of CE-MS, which mainly include the following. (1) High-performance capillary electrophoresis (HPCE) separation for minute amounts of tryptic peptide samples. Capillary electrochromatography can further improve the column capacity limit of HPCE. (2) CE-MS interfaces for high-sensitivity proteomics. Although sheath liquid interfaces have proven versatile and robust and are currently more commonly used, sheathless interfaces can significantly enhance the signal/noise ratio owing to decreased analyte dilution and background noise. Thus, sheathless interfaces are potentially more suitable for ultrasensitive proteomics. (3) Synergetic utilization of HPCE separation and MS detection at high scan rates. The most promising way to fully utilize the ultrahigh scan rates of modern high-resolution MS is to enhance the quality of peptide separation. Narrower peptide peaks in HPCE separation may greatly reduce redundant sampling and boost sensitivity. Overall, we anticipate that, after further improvement, CE-MS-based proteomics will be more widely applied to proteomic analysis of minute amounts of biological samples, such as single mammalian cells. Furthermore, more sensitive data acquisition modes, such as data-independent acquisition, may be used for global proteomic profiling, and parallel reaction monitoring may be used to target a limited number of important proteins. Matching between runs and machine learning algorithms may improve the accuracy of proteomic analysis of minute amounts of samples. Chemistry ; Capillary electrophoresis ; Chromatography ; Proteomics ; Sensitivity (control systems) ; Biochemistry Advanced Proteomics Techniques and Applications ; Bacteriophages and microbial interactions ; Genetics, Bioinformatics, and Biomedical Research