The development of selected reaction monitoring methods for targeted proteomics via empirical refinement

Bereman, Michael S.; MacLean, Brendan; Tomazela, Daniela M.; Liebler, Daniel C.; MacCoss, Michael J.

doi:10.1002/pmic.201200042

Cited by 103 publications

(88 citation statements)

References 55 publications

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“…The result files of the MaxQuant search obtained from the shotgun analyses were used to build an MS/MS library in Skyline, version 2.5 [21,22]. Besides the proteins selected for quality control assessment, as described in the BResultsŝ ection, 31 regulated proteins were considered for MRM analysis.…”

Section: Mrm Assay Developmentmentioning

confidence: 99%

Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

et al. 2015

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Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low abundant molecular species still rely on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study, we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama ( www.panoramaweb.org ). Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses, we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005 %, the accuracy was between 80 and 120 %, and the median coefficient of variation for LC/MS was only 2.2 %. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20 % for most peptides. With appropriate marker molecules, we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states, and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls. Graphical Abstract Work flow diagram: Data processing steps beginning with orbitrap-based shotgun data acquisition and MaxQuant data analysis, followed by peptide and transition selection for MRM analysis using Skyline and experimental validation using triple quadrupole MS.

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Section: Mrm Assay Developmentmentioning

confidence: 99%

Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

et al. 2015

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“…For the QTRAP 5500 all transitions were selected to be below m/z 1,250 due to the instrument's upper mass limit of m/z 1250 in Q1 and Q3. For the ThermoFisher Scientific and Agilent platforms, optimal peptides and their corresponding transitions were determined by predicting all tryptic peptides from the 6 ProteinMix-QC and by monitoring all y-ions from y 3 to y n-1 on the triple quadrupole TSQ Quantum-Ultra and Agilent 6460 (29). The raw data were imported into Skyline and results were refined to select the 5 most abundant transitions for all detectable peptides.…”

Section: Methodsmentioning

confidence: 99%

“…Skyline was used to build spectral libraries from data dependent acquisitions and peptide search engine results using the BiblioSpec library builder (28) so that MRM transitions could be selected based on previously acquired discovery platform data (for AB Sciex and Waters instruments). Alternatively (for Thermo and Agilent instruments), SRM Refinement approaches (29) were used on a triple quadrupole MS to determine optimal MRM transitions for each peptide.…”

Section: Methodsmentioning

confidence: 99%

“…Graphical Methods-Data from MRM experiments exported from Skyline and analyzed using the custom R code described above are further explored using a variety of plots, also generated using the R statistical computing environment (http://www.R-project.org) (29), specifically the Lattice Graphics package (34). Comprehensive plots were made to analyze not only the overall statistics such as CV, but also understand more detailed variation of the LC and MS parameters and measurements from replicate to replicate, between sites and across peptides.…”

Section: • Combine Information From All Five Transitions For Each Pepmentioning

confidence: 99%

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Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)

Abbatiello

Mani

Schilling

et al. 2013

Molecular & Cellular Proteomics

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105

101

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Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological andclinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system From the ‡Broad Institute of MIT and Harvard,

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“…A variety of methods have been previously used to identify signature peptides for protein quantification (2,15,16 ). One common approach is to target peptides identified by a data-dependent MS screen on related samples, as these peptides are guaranteed to be detectable by MS (2,17 ).…”

confidence: 99%

An Empirical Approach to Signature Peptide Choice for Selected Reaction Monitoring: Quantification of Uromodulin in Urine

Grote

Zhu

et al. 2016

Clinical Chemistry

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BACKGROUND: Many avenues have been proposed for a seamless transition between biomarker discovery data and selected reaction monitoring (SRM) assays for biomarker validation. Unfortunately, studies with the abundant urinary protein uromodulin have shown that these methods do not converge on a consistent set of surrogate peptides for targeted mass spectrometry. As an alternative, we present an empirical peptide selection work flow for robust protein quantification.

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The development of selected reaction monitoring methods for targeted proteomics via empirical refinement

Cited by 103 publications

References 55 publications

Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)

An Empirical Approach to Signature Peptide Choice for Selected Reaction Monitoring: Quantification of Uromodulin in Urine

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