10 research outputs found
Phosphoprotein secretome of tumor cells as a source of candidates for breast cancer biomarkers in plasma.
Breast cancer is a heterogeneous disease whose molecular diversity is not well reflected in clinical and pathological markers used for prognosis and treatment selection. As tumor cells secrete proteins into the extracellular environment, some of these proteins reach circulation and could become suitable biomarkers for improving diagnosis or monitoring response to treatment. As many signaling pathways and interaction networks are altered in cancerous tissues by protein phosphorylation, changes in the secretory phosphoproteome of cancer tissues could reflect both disease progression and subtype. To test this hypothesis, we compared the phosphopeptide-enriched fractions obtained from proteins secreted into conditioned media (CM) derived from five luminal and five basal type breast cancer cell lines using label-free quantitative mass spectrometry. Altogether over 5000 phosphosites derived from 1756 phosphoproteins were identified, several of which have the potential to qualify as phosphopeptide plasma biomarker candidates for the more aggressive basal and also the luminal-type breast cancers. The analysis of phosphopeptides from breast cancer patient plasma and controls allowed us to construct a discovery list of phosphosites under rigorous collection conditions, and second to qualify discovery candidates generated from the CM studies. Indeed, a set of basal-specific phosphorylation CM site candidates derived from IBP3, CD44, OPN, FSTL3, LAMB1, and STC2, and luminal-specific candidates derived from CYTC and IBP5 were selected and, based on their presence in plasma, quantified across all cell line CM samples using Skyline MS1 intensity data. Together, this approach allowed us to assemble a set of novel cancer subtype specific phosphopeptide candidates for subsequent biomarker verification and clinical validation
Multiplexed, Scheduled, High-Resolution Parallel Reaction Monitoring on a Full Scan QqTOF Instrument with Integrated Data-Dependent and Targeted Mass Spectrometric Workflows
Recent advances in commercial mass
spectrometers with higher resolving
power and faster scanning capabilities have expanded their functionality
beyond traditional data-dependent acquisition (DDA) to targeted proteomics
with higher precision and multiplexing. Using an orthogonal quadrupole
time-of flight (QqTOF) LC-MS system, we investigated the feasibility
of implementing large-scale targeted quantitative assays using scheduled,
high resolution multiple reaction monitoring (sMRM-HR), also referred
to as parallel reaction monitoring (sPRM). We assessed the selectivity
and reproducibility of PRM, also referred to as parallel reaction
monitoring, by measuring standard peptide concentration curves and
system suitability assays. By evaluating up to 500 peptides in a single
assay, the robustness and accuracy of PRM assays were compared to
traditional SRM workflows on triple quadrupole instruments. The high
resolution and high mass accuracy of the full scan MS/MS spectra resulted
in sufficient selectivity to monitor 6–10 MS/MS fragment ions
per target precursor, providing flexibility in postacquisition assay
refinement and optimization. The general applicability of the sPRM
workflow was assessed in complex biological samples by first targeting
532 peptide precursor ions in a yeast lysate, and then 466 peptide
precursors from a previously generated candidate list of differentially
expressed proteins in whole cell lysates from <i>E. coli</i>. Lastly, we found that sPRM assays could be rapidly and efficiently
developed in Skyline from DDA libraries when acquired on the same
QqTOF platform, greatly facilitating their successful implementation.
These results establish a robust sPRM workflow on a QqTOF platform
to rapidly transition from discovery analysis to highly multiplexed,
targeted peptide quantitation
SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks
SummaryReversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2
MS1 Peptide Ion Intensity Chromatograms in MS2 (SWATH) Data Independent Acquisitions. Improving Post Acquisition Analysis of Proteomic Experiments
SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target
Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation
Additional file 4 of Mapping age- and sex-specific HIV prevalence in adults in sub-Saharan Africa, 2000–2018
Additional file 4: Supplemental results.1. README. 2. Prevalence range across districts. 3. Prevalence range between sexes. 4. Prevalence range between ages. 5. Age-specific district ranges