Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics

Patil, Leena M.; Parkinson, David H; Zuniga, Nathan; Lin, Hsien-Jung L; Naylor, Bradley C.; Price, John C.

doi:10.1016/j.chroma.2023.464044

Cited by 3 publications

(3 citation statements)

References 37 publications

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“…High performance liquid chromatography (HPLC) off-line peptide separation (PS) technique has been used to enhance the detection of proteins by liquid chromatograph-tandem mass spectrometry (LC-MS /MS) and is a more effective method to improve the coverage of MS proteome [ 17 ]. The dried peptides were reconstituted with high-pH reverse-phase HPLC solution A (2% ACN, pH 10) and fractionated by using a C18 column (Waters BEH C18 4.6 × 250 mm, 5 μm) on a Shimadzu LC 15 HPLC operating at 0.6 mL/min.…”

Section: Methodsmentioning

confidence: 99%

Comparative proteomics reveals that fatty acid metabolism is involved in myocardial adaptation to chronic hypoxic injury

Chen,

Yu,

Zhang

et al. 2024

PLoS ONE

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Congenital heart disease (CHD) is the most serious form of heart disease, and chronic hypoxia is the basic physiological process underlying CHD. Some patients with CHD do not undergo surgery, and thus, they remain susceptible to chronic hypoxia, suggesting that some protective mechanism might exist in CHD patients. However, the mechanism underlying myocardial adaptation to chronic hypoxia remains unclear. Proteomics was used to identify the differentially expressed proteins in cardiomyocytes cultured under hypoxia for different durations. Western blotting assays were used to verify protein expression. A Real-Time Cell Analyzer (RTCA) was used to analyze cell growth. In this study, 3881 proteins were identified by proteomics. Subsequent bioinformatics analysis revealed that proteins were enriched in regulating oxidoreductase activity. Functional similarity cluster analyses showed that chronic hypoxia resulted in proteins enrichment in the mitochondrial metabolic pathway. Further KEGG analyses found that the proteins involved in fatty acid metabolism, the TCA cycle and oxidative phosphorylation were markedly upregulated. Moreover, knockdown of CPT1A or ECI1, which is critical for fatty acid degradation, suppressed the growth of cardiomyocytes under chronic hypoxia. The results of our study revealed that chronic hypoxia activates fatty acid metabolism to maintain the growth of cardiomyocytes.

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Section: Methodsmentioning

confidence: 99%

Comparative proteomics reveals that fatty acid metabolism is involved in myocardial adaptation to chronic hypoxic injury

Chen,

Yu,

Zhang

et al. 2024

PLoS ONE

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“…For discovery-based LC−MS/MS analysis, large cohorts of plasma samples are prepared with an extensive proteomic sample preparation workflow that can include (1) protein depletion, (2) protein digestion, (3) isotopic or isobaric labeling, 21,22 and (4) peptide fractionation. 23 Ideally, sample preparation workflows should be designed to minimize sample handling and operator-generated biases. However, sample preparation can be tedious, errorprone, and susceptible to reproducibility issues.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Plasma sample preparation steps can be tailored for the desired discovery or targeted proteomic analyses. For discovery-based LC–MS/MS analysis, large cohorts of plasma samples are prepared with an extensive proteomic sample preparation workflow that can include (1) protein depletion, (2) protein digestion, (3) isotopic or isobaric labeling, , and (4) peptide fractionation . Ideally, sample preparation workflows should be designed to minimize sample handling and operator-generated biases.…”

Section: Introductionmentioning

confidence: 99%

Establishing Quality Control Metrics for Large-Scale Plasma Proteomic Sample Preparation

Oliver,

Choi,

Arul

et al. 2024

ACS Meas. Sci. Au

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Large-scale plasma proteomics studies have been transformed due to the multiplexing and automation of sample preparation workflows. However, these workflows can suffer from reproducibility issues, a lack of standardized quality control (QC) metrics, and the assessment of variation before liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. The incorporation of robust QC metrics in sample preparation workflows ensures better reproducibility, lower assay variation, and better-informed decisions for troubleshooting. Our laboratory conducted a plasma proteomics study of a cohort of patient samples (N = 808) using tandem mass tag (TMT) 16-plex batches (N = 58). The proteomic workflow consisted of protein depletion, protein digestion, TMT labeling, and fractionation. Five QC sample types (QCstd, QCdig, QCpool, QCTMT, and QCBSA) were created to measure the performance of sample preparation prior to the final LC–MS/MS analysis. We measured <10% CV for individual sample preparation steps in the proteomic workflow based on data from various QC sample steps. The establishment of robust measures for QC of sample preparation steps allowed for greater confidence in prepared samples for subsequent LC–MS/MS analysis. This study also provides recommendations for standardized QC metrics that can assist with future large-scale cohort sample preparation workflows.

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Preparation of Halloysite Nanotube-Based Monolithic Column for Protein Analysis

Zhao,

Guo,

et al. 2024

Preprint

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Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics

Cited by 3 publications

References 37 publications

Comparative proteomics reveals that fatty acid metabolism is involved in myocardial adaptation to chronic hypoxic injury

Comparative proteomics reveals that fatty acid metabolism is involved in myocardial adaptation to chronic hypoxic injury

Establishing Quality Control Metrics for Large-Scale Plasma Proteomic Sample Preparation

Preparation of Halloysite Nanotube-Based Monolithic Column for Protein Analysis

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