The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses.

Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.

Narrow mass extraction of time-of-flight data for quantitative analysis of proteins: determination of insulin-like growth factor-1.

Methods for quantitative analysis of proteins by mass spectrometry have progressed dramatically. While isotope-dilution approaches using selected reaction monitoring of tryptic peptides (also known as bottom up) have become common, the potential to use narrow mass extraction of high-resolution mass spectra provides a compelling alternative. We investigated the relationships between instrument performance and data processing with the aim of determining whether this approach can lead to robust bioanalytical assays for proteins. Our approach utilized off-line sample preparation combined with online sample extraction coupled to HPLC with the effluent from the analytical column directed to a high-resolution, high-mass accuracy quadrupole time-of-flight (qTOF) mass spectrometer operated in full scan mode. Narrow mass extraction of a single isotope from IGF-1 in the 7+ charge state (m/z 1093.5209) was used to generate extracted ion chromatograms. We found that with appropriate attention to instrument performance and data processing, quantitative protein assays with good sensitivity, high selectivity, and excellent analytical performance can be developed.

Plasma renin activity by LC-MS/MS: development of a prototypical clinical assay reveals a subpopulation of human plasma samples with substantial peptidase activity.

BACKGROUND: For management and treatment of secondary hypertension, plasma renin activity (PRA) assay is considered an essential diagnostic tool. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to PRA offering improvements in laboratory workflow and throughput. During development, we observed a substantial number of clinical samples that have strong degradation activity toward angiotensin (Ang) I during generation. A preliminary characterization of this degradation activity was performed, and we provide here a method by which this degradation can be monitored via the addition of an isotope-labeled degradation standard. METHODS: Automated online sample extraction coupled with HPLC was used to isolate Ang I and internal standard from plasma. The effluent from the analytical column was directed to a triple quadrupole MS operated in selected reaction monitoring mode, monitoring the a(5) and b(5) product ions from the [M+3H](+3) precursors. Routine analysis could be achieved with as little as 150 µL plasma. RESULTS: We identified both C-terminal and N-terminal degradation products of Ang I using isotope-labeled peptides as controls and substrates. In 2%-5% of patient samples, the degradation essentially eliminated any Ang I produced during generation. CONCLUSIONS: Our method requires reduced sample handling when compared with an RIA and eliminates the need for extended generation times for samples with low renin activity. Degradation of Ang I during generation appears to be a confounding variable in the interpretation of results from some clinical samples. Samples with profound degradation activity can be identified using a degradation standard that is added at the start of generation.