Use of monolithic supports for high‐throughput protein and peptide separation in proteomics

Abstract

The exclusive properties of monolithic supports enable fast mass transfer, high porosity, low back pressure, easy preparation process and miniaturisation, and the availability of different chemistries make them particularly suitable materials for high-throughput (HTP) protein and peptide separation. In this review recent advances in monolith-based chromatographic supports for HTP screening of protein and peptide samples are presented and their application in HTP sample preparation (separation, enrichment, depletion, proteolytic digestion) for HTP proteomics is discussed. Development and applications of different monolithic capillary columns in HTP MS-based bottom-up and top-down proteomics are overviewed. By discussing the chromatographic conditions and the mass spectrometric data acquisition conditions an attempt is made to present currently demonstrated capacities of monolithic capillary columns for HTP identification and quantification of proteins and peptides from complex biological samples by MS-based proteomics. Some recent advances in basic monolith technology of importance for proteomics are also discussed.

A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field

“…Miniaturized HPLC separation systems have been developed by using fused silica capillary columns [96,97] or chip-based devices [98][99][100]. Monolithic capillary columns (and other columns, such as C8 or C18 columns) can be produced in the laboratory without the need of expensive media, packing solvents, and high-pressure packing instrumentation with the proper training [101]. Even if reproducibility issues of published protocols to produce such columns were reported, they remain cost-efficient and depend on the researcher's capability to pack it perfectly.…”

Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry

“…The optimization of the LC gradient for the non-14.1 kDa ASVs is expected to improve the number of identifications with the current resin, but new SPLC resins as well as alternative resins (e.g., monoliths, 49,50 submicron silica particles 51,52 ) or LC strategies (e.g., IEC, HILIC, and UPLC) 53 are expected to provide enhanced separations and extend the platform to much larger, heavily modified proteins.…”

Challenges and Advances in the Fabrication of Monolithic Bioseparation Materials and their Applications in Proteomics Research

“…For the sake of improvement of peak capacity, long columns may be used and packed with smaller particles, which however would lead to a higher back pressure over 70 MPa in ultrahigh performance liquid chromatography (UPLC) system . As a good alternative to particle‐packed columns, monolithic columns exhibiting higher permeability and faster mass transfer are applied in either nano‐LC or cLC to increase the sensitivity in proteome analysis . Recently, there are several review articles focusing on the development of monolithic columns for chromatographic analysis of biomolecules .…”