Advances in microscale separations towards nanoproteomics applications

Abstract

Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. In recent decades significant advances have been achieved in MS-based proteomics. However, the current proteomics platforms still face an analytical challenge in overall sensitivity towards nanoproteomics applications for starting materials of less than 1μg total proteins (e.g., cellular heterogeneity in tissue pathologies). Herein, we review recent advances in microscale separation techniques and integrated sample processing strategies that improve the overall sensitivity and proteome coverage of the proteomics workflow, and their contributions towards nanoproteomics applications.

Keywords: Capillary electrophoresis; Mass spectrometry; Microscale separations; NanoLC; Nanoproteomics.

Fig. 1.

An illustration of traditional and nanoproteomics domains. The nanoproteomics is defined for dealing with samples containing <1 μg total protein in starting material.

Fig. 2.

A general workflow of bottom-up MS-based proteomics. It typically starts from cell lysis, protein extraction, and proteolytic digestion to peptide mixtures, followed up by fractionation or enrichment procedures and LC or CE-MS/MS analyses. The experimental spectrum is matched with database to identify peptides or contaminants.

Fig. 3.

An overview of techniques adopted in different components of MS-based nanoproteomics. Generally, nanogram-scale samples are obtained from different sources, and are processed using microscale sample processing techniques such as single-tube, or online processing systems to reduce sample transfer steps for minimum sample loss. Then the digested peptides are subjected to highly sensitive nanoLC-MS or CE-MS analysis. In some cases, multidimensional separation is utilized to increase overall proteome coverage.

Fig. 4.

Schematics of online integrated sample processing techniques. (A) SNaPP system for global proteomics of nanogram samples. Adpated from Ref [91]. (B) A monolithic column was used as a proteomic reactor and then assembled as the first dimensional SCX separation column. Adapted from Ref [95]. (C) iPAD system for living cell proteome profiling. Adapted with permission from Ref [100]. Copyright (2015) American Chemical Society.