Unknown

Dataset Information

0

A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples


ABSTRACT: We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30–60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mass spectrometry approaches and to in-depth proteomic studies where the amount of sample is limited. Using targeted proteomics with peptide standards, we present the method's analytical parameters, and potential in increasing the detection of low-abundance proteins that are difficult to quantify with direct targeted or global LC-MS analyses. This fractionation system increased peptide signals by up to 18-fold, while maintaining high quantitative precision, with high fractionation reproducibility across varied sample sets. In real applications, it increased the detection of targeted endogenous peptides by two-fold in a 25 cell-cycle-control protein panel, and in-depth MS analyses of nuclear extracts, it allowed the detection of up to 8,896 proteins with 138,417 peptides in 24-concatenated fractions compared to 3,344 proteins with 23,093 peptides without fractionation. In a relevant biological problem of CDK4/6-inhibitors and breast cancer, the method reproduced known information and revealed novel insights, highlighting that it can be successfully applied in studies involving low-abundance proteins and limited samples. • Tested nine high-pH buffer/solvent systems to obtain a robust, effective, and reproducible micro-flow fractionation method which was devoid of commonly encountered LC clogging/pressure issues after months of use.• Peptide enrichment method to improve detection and quantitation of low-abundance proteins in targeted and in-depth proteomic studies.• Can be applied to diverse protein samples where the available amount is limited. Graphical abstract Image, graphical abstract

SUBMITTER: Zurawska M 

PROVIDER: S-EPMC10413349 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

Similar Datasets

2014-10-23 | PXD001404 | Pride
| S-EPMC3385641 | biostudies-literature
| S-EPMC6516780 | biostudies-literature
| S-EPMC7312998 | biostudies-literature
| S-EPMC7544661 | biostudies-literature
| S-EPMC9751300 | biostudies-literature
| S-EPMC6741344 | biostudies-literature
| S-EPMC11256012 | biostudies-literature
| S-EPMC11923864 | biostudies-literature
| S-EPMC8317427 | biostudies-literature