A Mini Review on Capillary Isoelectric Focusing-Mass Spectrometry for Top-Down Proteomics

Abstract

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.

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

“…Capillary isoelectric focusing (cIEF) is a powerful electrophoretic technique for proteoform and even protein complex separations based on their isoelectric points (pIs) with extremely high resolution. − It has a drastically higher sample loading capacity (maximum loading of entire capillary volume) compared to CZE (typical loading of less than 1% of the capillary volume). In addition, cIEF can measure the pIs of proteoforms accurately using pI standards .…”

Applying multi‐omics toward tumor microbiome research

“…There are three different strategies of proteomic analysis: "topdown," "middle-down," and "bottom-up." Specifically, "top-down" (Figure 4A) [142] directly sends target proteins to mass spectrometry (MS) for analysis, without any pretreatment. "Middle-down" [143] refers to proteins being partially digested to obtain relatively large peptide fragments, before being sent to MS for further analysis.…”

Raman Spectroscopy—A Novel Method for Identification and Characterization of Microbes on a Single-Cell Level in Clinical Settings

“…These approaches include but are not limited to certain protocols for MALDI-TOF MS (Drancourt, 2010;Meex et al, 2012;Hoyos-Mallecot et al, 2014;Idelevich et al, 2014;Machen et al, 2014;Verroken et al, 2015;Zhou et al, 2017), infrared spectroscopy (FTIR) (Ojeda and Dittrich, 2012;Zarnowiec et al, 2015;Vogt et al, 2019), nuclear magnetic resonance (NMR) spectroscopy (Romaniuk and Cegelski, 2015;Palama et al, 2016), capillary electrophoresis (incl. capillary isoelectric focusing (Ruzicka et al, 2016;Xu and Sun, 2021), electrical field-flow fractionation (Saenton et al, 2000;Reschiglian et al, 2002), microfluidic devices (Kim et al, 2012;Zhou et al, 2019;Peŕez-Rodrıǵuez et al, 2022) and Raman spectroscopy (Franco-Duarte et al, 2019). Recently, Raman spectroscopy has been undergoing a boom in microbiology, as many studies suggest its potential for the identification of microbes, their virulence factors, detection of metabolic changes, and last but not least, single-cell analyses of microbial cells.…”