Facile Preparation of Peptides for Mass Spectrometry Analysis in Bottom‐Up Proteomics Workflows

Abstract

Mass spectrometry (MS) is routinely used to identify, characterize, and quantify biological molecules. For protein analysis, MS-based workflows can be broadly categorized as top-down or bottom-up, depending on whether the proteins are analyzed as intact molecules or first digested into peptides. This article outlines steps for preparing peptide samples for MS as part of a bottom-up proteomics workflow, providing versatile methods suitable for discovery and targeted analyses in qualitative and quantitative workflows. Resulting samples contain peptides of suitable size for analysis by MS instrumentation generally available to modern research laboratories, including MS coupled to either liquid chromatography (LC) or matrix-assisted laser desorption/ionization (MALDI) interfaces. This article incorporates recent developments in methodologies and consumables to facilitate sample preparation. The protocols are well-suited to users without prior experience in proteomics and include methods for universally applicable suspension trap processing and for alternate in-solution processing to accommodate a range of sample types. Cleanup, quantification, and fractionation procedures are also described.

Novel Broccoli-Derived Peptides Hydrolyzed by Trypsin with Dual-Angiotensin I-Converting Enzymes and Dipeptidyl Peptidase-IV-Inhibitory Activities

“…20 μL of each standard (Peptide Digest Assay Standard, supplied by the assay kit) or unknown sample was used and incubated at 22 °C (room temperature) for 30 min, and the detection wavelength was 480 nm. The peptide concentration was counted from the calibration curve of the standard sample …”

Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes

“…This fast and easy approach to processing bottom-up proteomic samples for MS analysis does not require specialized expertise. A single experimenter can typically generate MS-ready peptide samples in 8 h using this method, including sample homogenization, lysis, and digestion [ 22 ]. As part of reproducibility assessment, dot plots of the number of peptide groups found in individual samples of each unfractionated workflows (II and IV) were assessed ( Fig.…”

“…To facilitate implementation of these approaches, detailed methods and reagent information are in the supplementary information . Protein digestion and peptide fractionation methods are based on detailed protocols we recently described [ 22 ].…”

Proteomic Analysis of Adenovirus 5 by UHPLC-MS/MS: Development of a Robust and Reproducible Sample Preparation Workflow

“…There have been strenuous efforts to develop a universal, robust, and reproducible sample processing strategy for proteomic analysis. − Important factors to consider when choosing procedures to minimize sample losses and maximize detection of peptides by MS include sample concentration and matrix type. However, there is little consensus regarding optimal sample processing protocols, and this step remains the main bottleneck for successful proteomic analysis.…”