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Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.

ABSTRACT: Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this "fit-for-purpose" approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.

SUBMITTER: Carr SA

PROVIDER: S-EPMC3945918 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Mass spectrometry (MS) is a very sensitive and specific method for protein identification, biomarker discovery, and biomarker validation. Protein identification is commonly carried out by comparing MS data with public databases. However, with the development of high throughput and accurate genomic sequencing technology, public databases are being overwhelmed with new entries from different species every day. The application of these databases can also be problematic due to factors such as size, specificity, and unharmonized annotation of the molecules of interest. Current databases representing liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based searches focus on enzyme digestion patterns and sequence information and consequently, important functional information can be missed within the search output. Protein variants displaying similar sequence homology can interfere with database identification when only certain homologues are examined. In addition, recombinant DNA technology can result in products that may not be accurately annotated in public databases. Curated databases, which focus on the molecule of interest with clearer functional annotation and sequence information, are necessary for accurate protein identification and validation. Here, four cases of curated database application have been explored and summarized. FINDINGS: The four presented curated databases were constructed with clear goals regarding application and have proven very useful for targeted protein identification and biomarker application in different fields. They include a sheeppox virus database created for accurate identification of proteins with strong antigenicity, a custom database containing clearly annotated protein variants such as tau transcript variant 2 for accurate biomarker identification, a sheep-hamster chimeric prion protein (PrP) database constructed for assay development of prion diseases, and a custom Escherichia coli (E. coli) flagella (H antigen) database produced for MS-H, a new H-typing technique. Clearly annotating the proteins of interest was essential for highly accurate, specific, and sensitive sequence identification, and searching against public databases resulted in inaccurate identification of the sequence of interest, while combining the curated database with a public database reduced both the confidence and sequence coverage of the protein search. CONCLUSION: Curated protein sequence databases incorporating clear annotations are very useful for accurate protein identification and fit-for-purpose application through MS-based biomarker validation.

Project description:BackgroundAs many as 90% of patients develop anemia by their third day in an intensive care unit (ICU). We evaluated the efficacy of interventions to reduce phlebotomy-related blood loss on the volume of blood lost, hemoglobin levels, transfusions, and incidence of anemia.MethodsWe conducted a systematic review and meta-analysis using the Laboratory Medicine Best Practices (LMBP) systematic review methods for rating study quality and assessing the body of evidence. Searches of PubMed, Embase, Cochrane, Web of Science, PsychINFO, and CINAHL identified 2564 published references. We included studies of the impact of interventions to reduce phlebotomy-related blood loss on blood loss, hemoglobin levels, transfusions, or anemia among hospital inpatients. We excluded studies not published in English and studies that did not have a comparison group, did not report an outcome of interest, or were rated as poor quality. Twenty-one studies met these criteria. We conducted a meta-analysis if > 2 homogenous studies reported sufficient information for analysis.ResultsWe found moderate, consistent evidence that devices that return blood from flushing venous or arterial lines to the patient reduced blood loss by approximately 25% in both neonatal ICU (NICU) and adult ICU patients [pooled estimate in adults, 24.7 (95% CI = 12.1-37.3)]. Bundled interventions that included blood conservation devices appeared to reduce blood loss by at least 25% (suggestive evidence). The evidence was insufficient to determine if these devices reduced hemoglobin decline or risk of anemia. The evidence suggested that small volume tubes reduced the risk of anemia, but was insufficient to determine if they affected the volume of blood loss or the rate of hemoglobin decline.ConclusionsModerate, consistent evidence indicated that devices that return blood from testing or flushing lines to the patient reduce the volume of blood loss by approximately 25% among ICU patients. The results of this systematic review support the use of blood conservation systems with arterial or venous catheters to eliminate blood waste when drawing blood for testing. The evidence was insufficient to conclude the devices impacted hemoglobin levels or transfusion rates. The use of small volume tubes may reduce the risk of anemia.

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Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.

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