A Proteomics Tutorial

Merkley, Eric; Kaiser, Brooke L. Deatherage; Kreuzer, Helen

doi:10.1021/bk-2019-1339.ch002

Cited by 4 publications

(2 citation statements)

References 130 publications

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“…Overall, in the bottom-up workflow, proteins undergo enzymatic digestion by a protease, and the sizes of proteins are reduced from hundreds of amino acids to peptides with six to 30 amino acids. Therefore, at first, the specific protein is separated by the LC, then it is fragmented by enzymatic digestion, and then finally coupled to MS [67,68]. When the bottom-up methods are employed on the complex protein sample (such as proteome), the untargeted procedure is termed as shotgun proteomics [69].…”

Section: Based On the Study Aimsmentioning

confidence: 99%

Proteomics techniques in protein biomarker discovery

Babaei,

Kashanian,

Lee

et al. 2024

Quant. Biol.

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Protein biomarkers represent specific biological activities and processes, so they have had a critical role in cancer diagnosis and medical care for more than 50 years. With the recent improvement in proteomics technologies, thousands of protein biomarker candidates have been developed for diverse disease states. Studies have used different types of samples for proteomics diagnosis. Samples were pretreated with appropriate techniques to increase the selectivity and sensitivity of the downstream analysis and purified to remove the contaminants. The purified samples were analyzed by several principal proteomics techniques to identify the specific protein. In this study, recent improvements in protein biomarker discovery, verification, and validation are investigated. Furthermore, the advantages, and disadvantages of conventional techniques, are discussed. Studies have used mass spectroscopy (MS) as a critical technique in the identification and quantification of candidate biomarkers. Nevertheless, after protein biomarker discovery, verification and validation have been required to reduce the false‐positive rate where there have been higher number of samples. Multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), and selected reaction monitoring (SRM), in combination with stable isotope‐labeled internal standards, have been examined as options for biomarker verification, and enzyme‐linked immunosorbent assay (ELISA) for validation.

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Section: Based On the Study Aimsmentioning

confidence: 99%

Proteomics techniques in protein biomarker discovery

Babaei,

Kashanian,

Lee

et al. 2024

Quant. Biol.

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“…Generally, sample preparation aims to create a less complex peptide mixture that is suitable for analysis. It requires pre-fractionation; depletion of most unnecessarily abundant proteins; removal of DNA, lipids, and small metabolites; and sample clean-up from impurities (salts and remaining solid particles) [ 51 ]. Therefore, typical sample preparation processes for bottom-up proteomics usually include lysis/homogenization, protein extraction/precipitation, reduction, alkylation, enzymatic digestion, fractionation, and desalting ( Figure 1 ).…”

Section: Introductionmentioning

confidence: 99%

Bottom-Up Proteomics: Advancements in Sample Preparation

Duong

Lee

2023

IJMS

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Liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomics is a powerful technique for profiling proteomes of cells, tissues, and body fluids. Typical bottom-up proteomic workflows consist of the following three major steps: sample preparation, LC–MS/MS analysis, and data analysis. LC–MS/MS and data analysis techniques have been intensively developed, whereas sample preparation, a laborious process, remains a difficult task and the main challenge in different applications. Sample preparation is a crucial stage that affects the overall efficiency of a proteomic study; however, it is prone to errors and has low reproducibility and throughput. In-solution digestion and filter-aided sample preparation are the typical and widely used methods. In the past decade, novel methods to improve and facilitate the entire sample preparation process or integrate sample preparation and fractionation have been reported to reduce time, increase throughput, and improve reproducibility. In this review, we have outlined the current methods used for sample preparation in proteomics, including on-membrane digestion, bead-based digestion, immobilized enzymatic digestion, and suspension trapping. Additionally, we have summarized and discussed current devices and methods for integrating different steps of sample preparation and peptide fractionation.

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