Flash entropy search to query all mass spectral libraries in real time

Nature Methods, Journal Year: 2023, Volume and Issue: 20(10), P. 1475 - 1478

Published: Sept. 21, 2023

Language: Английский

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A microfluidics-enabled automated workflow of sample preparation for MS-based immunopeptidomics

Cell Reports Methods, Journal Year: 2023, Volume and Issue: 3(6), P. 100479 - 100479

Published: May 19, 2023

Mass spectrometry (MS)-based immunopeptidomics is an attractive antigen discovery method with growing clinical implications. However, the current experimental approach to extract HLA-restricted peptides requires a bulky sample source, which remains challenge for obtaining specimens. We present innovative workflow that low volume, streamlines immunoaffinity purification (IP) and C18 peptide cleanup on single microfluidics platform automated liquid handling minimal transfers, resulting in higher assay sensitivity. also demonstrate how state-of-the-art data-independent acquisition (DIA) further enhances depth of tandem MS spectra-based sequencing. Consequently, over 4,000 5,000 HLA-I-restricted were identified from as few 0.2 million RA957 cells melanoma tissue merely 5 mg, respectively. multiple immunogenic tumor-associated antigens hundreds derived non-canonical protein sources. This represents powerful tool identifying immunopeptidome sparse samples.

Language: Английский

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Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins

Journal of Proteome Research, Journal Year: 2024, Volume and Issue: 23(4), P. 1531 - 1543

Published: March 20, 2024

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes streptavidin resins influence PDB results. We compared high-confidence interactors DNA/RNA-binding transactive response DNA-binding 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. also evaluated two commercial affinity for purification biotinylated proteins: conventional sepharose versus new trypsin-resistant conjugated to magnetic resin, which significantly reduces level contamination by peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled mass spectrometry in data-dependent acquisition mode, database searching, statistical analysis SAINTexpress. The APEX2-TDP-43 experiment more than miniTurbo-TDP-43, although provided greater overlap with previously documented TDP-43 interactors. Purifications on resin yielded small-scale experiments range volumes. suggest that resin-specific background binding profiles lysate-to-resin ratios cumulatively affect distributions prey abundance experimental control samples, impact confidence scores. Overall, highlight key variables consider empirical optimization experiments.

Language: Английский

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High-Throughput Proteomics and Phosphoproteomics of Rat Tissues Using Microflow Zeno SWATH

Journal of Proteome Research, Journal Year: 2024, Volume and Issue: 23(7), P. 2355 - 2366

Published: May 31, 2024

High-throughput tissue proteomics has great potential in the advancement of precision medicine. Here, we investigated combined sensitivity trap-elute microflow liquid chromatography with a ZenoTOF for DIA and phosphoproteomics. Method optimization was conducted on HEK293T cell lines to determine optimal variable window size, MS2 accumulation time gradient length. The 7600 then compared previous generation TripleTOF 6600 using eight rat organs, finding up 23% more proteins fifth sample load third instrument time. Spectral reference libraries generated from Zeno SWATH data FragPipe (MSFragger-DIA/DIA-NN) contained 4 times fragment ions than DIA-NN only library quantified proteins. Replicate single-shot phosphopeptide enrichments 50–100 μg tryptic peptide were analyzed by HPLC without fractionation. Using Spectronaut shallow phosphoproteome containing 1000–3000 phosphoprecursors per organ. Promisingly, clear hierarchical clustering organs observed high Pearson correlation coefficients >0.95 between replicate median CV 20%. allows high-throughput quantitation an extensive proteome small samples.

Language: Английский

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Protein profiling of zebrafish embryos unmasks regulatory layers during early embryogenesis

Cell Reports, Journal Year: 2024, Volume and Issue: 43(10), P. 114769 - 114769

Published: Sept. 19, 2024

Language: Английский

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