Pipelines and Systems for Threshold-Avoiding Quantification of LC-MS/MS Data.
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ABSTRACT:
SUBMITTER: Sanchez Brotons A
PROVIDER: S-EPMC8374884 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The accurate processing of complex LC-MS/MS data from biological samples is a major challenge for metabolomics, proteomics and related approaches. Here we present the Pipelines and Systems for Threshold Avoiding Quantification (PASTAQ) LC-MS/MS pre-processing toolset, which allows highly accurate quantification of data-dependent acquisition (DDA) LC-MS/MS datasets. PASTAQ performs compound quantification using single-stage (MS1) data and implements novel algorithms for high-performance and accurate quantification, retention time alignment, feature detection, and linking annotations frommultiple identification engines. PASTAQ offers straightforward parametrization and automatic generation of quality control plots for data and pre-processing assessment. This design results in smaller variance when analyzing replicates of proteomes mixed with known ratios, and allows the detection of peptides with a larger dynamic concentration range compared to widely used proteomics preprocessing tools. The performance of the pipeline is also demonstrated in a biological human serum dataset for the identification of gender related proteins.
2021-08-18 | PXD024584 | Pride
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Project description:Data-independent acquisition LC-MS/MS techniques complement supervised methods for peptide quantification. However, due to the wide precursor isolation windows, these techniques are prone to interference at the fragment ion level, which, in turn, is detrimental for accurate quantification. The nonoutlier fragment ion (NOFI) ranking algorithm has been developed to assign low priority to fragment ions affected by interference. By using the optimal subset of high-priority fragment ions, these interfered fragment ions are effectively excluded from quantification. NOFI represents each fragment ion as a vector of four dimensions related to chromatographic and MS fragmentation attributes and applies multivariate outlier detection techniques. Benchmarking conducted on a well-defined quantitative data set (i.e., the SWATH Gold Standard) indicates that NOFI on average is able to accurately quantify 11-25% more peptides than the commonly used Top-N library intensity ranking method. The sum of the area of the Top3-5 NOFIs produces similar coefficients of variation as compared to that with the library intensity method but with more accurate quantification results. On a biologically relevant human dendritic cell digest data set, NOFI properly assigns low-priority ranks to 85% of annotated interferences, resulting in sensitivity values between 0.92 and 0.80, against 0.76 for the Spectronaut interference detection algorithm.
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Project description:We report a sensitive LC (liquid chromatography)/MS/MS assay using selected reaction monitoring to quantify RA (retinoic acid), which is applicable to biological samples of limited size (10-20 mg of tissue wet weight), requires no sample derivatization, provides mass identification and resolves atRA (all-trans-RA) from its geometric isomers. The assay quantifies over a linear range of 20 fmol to 10 pmol, and has a 10 fmol limit of detection at a signal/noise ratio of 3. Coefficients of variation are: instrumental, 0.5-2.9%; intra-assay, 5.4+/-0.4%; inter-assay 8.9+/-1.0%. An internal standard (all-trans-4,4-dimethyl-RA) improves accuracy by confirming extraction efficiency and revealing handling-induced isomerization. Tissues of 2-4-month-old C57BL/6 male mice had atRA concentrations of 7-9.6 pmol/g and serum atRA of 1.9+/-0.6 pmol/ml (+/-S.E.M.). Tissue 13-cis-RA ranged from 2.9 to 4.2 pmol/g, and serum 13-cis-RA was 1.2+/-0.3 pmol/ml. CRBP (cellular retinol-binding protein)-null mouse liver had atRA approximately 30% lower than wild-type (P<0.05), but kidney, testis, brain and serum atRA were similar to wild-type. atRA in brain areas of 12-month-old female C57BL/6 mice were (+/-S.E.M.): whole brain, 5.4+/-0.4 pmol/g; cerebellum, 10.7+/-0.3 pmol/g; cortex, 2.6+/-0.4 pmol/g; hippocampus, 8.4+/-1.2 pmol/g; striatum, 15.3+/-4.7 pmol/g. These data provide the first analytically robust quantification of atRA in animal brain and in CRBP-null mice. Direct measurements of endogenous RA should have a substantial impact on investigating target tissues of RA, mechanisms of RA action, and the relationship between RA and chronic disease.
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Project description:BACKGROUND:Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). METHODS:We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, (15)N-labeled recombinant HSA ((15)NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides (42)LVNEVTEFAK(51) and (526)QTALVELVK(534) from albumin and (15)NrHSA were referenced to the calibration curve. RESULTS:The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13-200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%-11.0% for the peptide (42)LVNEVTEFAK(51), and 1.9%-12.3% for the (526)QTALVELVK(534) peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%-14.8% for the (42)LVNEVTEFAK(51) peptide, and 6.4%-14.1% for the (526)QTALVELVK(534) peptide. For the (42)LVNEVTEFAK(51) peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R(2) value of 0.97 and a regression line of y = 0.99x + 23.16. CONCLUSIONS:Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a (15)NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.
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