We present an instrument-independent benchmark procedure and software (LFQ_bout) for the validation and comparative evaluation of the performance of LC-MS/MS and data processing workflows in bottom-up proteomics. The procedure enables a back-to-back comparison of common and emerging workflows, ., diaPASEF or ScanningSWATH, and evaluates the impact of arbitrary and inadequately documented settings or black-box data processing algorithms. It enhances the overall performance and quantification accuracy by recognizing and reporting common quantification errors.
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http://dx.doi.org/10.1021/acs.jproteome.3c00531 | DOI Listing |
J Proteome Res
January 2025
Advanced Research Support Center, Ehime University, Ehime 791-0295, Japan.
Precise prefractionation of proteome samples is a potent method for realizing in-depth analysis in top-down proteomics. PEPPI-MS (Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS), a gel-based sample fractionation method, enables high-resolution proteome fractionation based on molecular weight by highly efficient extraction of proteins from polyacrylamide gels after SDS-PAGE separation. Thereafter it is essential to effectively remove contaminants such as CBB and SDS from the PEPPI fraction prior to mass spectrometry.
View Article and Find Full Text PDFJ Proteome Res
January 2025
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, 69120 Heidelberg, Germany.
The first step in bottom-up proteomics is the assignment of measured fragmentation mass spectra to peptide sequences, also known as peptide spectrum matches. In recent years novel algorithms have pushed the assignment to new heights; unfortunately, different algorithms come with different strengths and weaknesses and choosing the appropriate algorithm poses a challenge for the user. Here we introduce PeptideForest, a semisupervised machine learning approach that integrates the assignments of multiple algorithms to train a random forest classifier to alleviate that issue.
View Article and Find Full Text PDFAnal Chim Acta
February 2025
Department of Chemistry, University of Texas at Austin, Austin, TX, 78712, United States. Electronic address:
Chemical proteomics has advanced small molecule ligand discovery by providing insights into protein-ligand binding mechanism and enabling medicinal chemistry optimization of protein selectivity on a global scale. Mass spectrometry is the predominant analytical method for chemoproteomics, and various approaches have been deployed to investigate and target a rapidly growing number of protein classes and biological systems. Two methods, intact mass analysis (IMA) and top-down proteomics (TDMS), have gained interest in recent years due to advancements in high resolution mass spectrometry instrumentation.
View Article and Find Full Text PDFAnal Chem
January 2025
Department of Electrical Engineering and Electronics, University of Liverpool, Liverpool L69 3BX, U.K.
Reports of proteins in fossilized bones have been a subject of controversy in the scientific literature because it is assumed that fossilization results in the destruction of all organic components. In this paper, a novel combination of analytical techniques is used to address this question for an exceptionally well-preserved sacrum excavated from the Upper Cretaceous strata of the South Dakota Hell Creek Formation. Cross-polarized light microscopy (XPol) shows birefringence consistent with collagen presence.
View Article and Find Full Text PDFNat Protoc
January 2025
Advanced Research Support Center, Ehime University, Ehime, Japan.
Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS-PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics.
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