Transitioning from Targeted to Comprehensive Mass

Authors: Jacob D Jaffe Caitlin M Feeney Jinal Patel Xiaodong Lu D R Mani

Publish Date: 2016/08/25

Volume: 27, Issue: 11, Pages: 1745-1751

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Abstract

Targeted proteomic assays are becoming increasingly popular because of their robust quantitative applications enabled by internal standardization and they can be routinely executed on high performance mass spectrometry instrumentation However these assays are typically limited to 100s of analytes per experiment Considerable time and effort are often expended in obtaining and preparing samples prior to targeted analyses It would be highly desirable to detect and quantify 1000s of analytes in such samples using comprehensive mass spectrometry techniques eg SWATH and DIA while retaining a high degree of quantitative rigor for analytes with matched internal standards Experimentally it is facile to port a targeted assay to a comprehensive data acquisition technique However data analysis challenges arise from this strategy concerning agreement of results from the targeted and comprehensive approaches Here we present the use of genetic algorithms to overcome these challenges in order to configure hybrid targeted/comprehensive MS assays The genetic algorithms are used to select precursortofragment transitions that maximize the agreement in quantification between the targeted and the comprehensive methods We find that the algorithm we used provided acrosstheboard improvement in the quantitative agreement between the targeted assay data and the hybrid comprehensive/targeted assay that we developed as measured by parameters of linear models fitted to the results We also found that the algorithm could perform at least as well as an independentlytrained mass spectrometrist in accomplishing this task We hope that this approach will be a useful tool in the development of quantitative approaches for comprehensive proteomics techniquesTargeted proteomic assays are becoming increasingly popular because of their robust quantitative applications enabled by introduction of stable isotopelabeled SIL internal standards reproducibility of analyte detection and ‘designability’ to answer specific questions or monitor biological processes 1 2 3 4 It is becoming common to execute these assays on high performance mass spectrometry instrumentation high resolution accurate mass in addition to the traditional triple quadrupole instrumentation When executed on high performance MS instruments targeted approaches record the full MS/MS spectrum of all fragment ions generated from a given dissociation technique The technique goes by various monikers such as parallel reaction monitoring PRM MRMHR multiple reaction monitoringhigh resolution HRMRM etc Use of full scan MS/MS spectra in targeted assays allows for selection of many interferencefree transitions for quantification and unambiguous identification of analytes including localization of sites of posttranslational modifications on peptides 5 Although perhaps not as sensitive as triple quadrupole instrumentation high resolution instrumentation allows for more selectivity and less potential for interferenceTargeted methods have been used extensively for biomarker verification studies 6 7 8 9 10 and are emerging for acquisition of assay panels with specific analytical purposes For example the emergence of “sentinel assays” to study the changes in specific biological processes or activation of signaling pathways demonstrates an increasing role for targeted proteomics in biology 4 11 12 In almost all cases great care is placed in the selection of samples and their associated biochemical preparation prior to analysis In some applications specific biochemical enrichments are performed to isolate special populations of analytes such as phosphopeptides Yet targeted analyses are typically limited to detection of only ~100s of analytes in a single assay of reasonable laboratory time scale ~1–2 h The practical limits are typically set by hardware duty cycle and scheduling requirements Deeper interrogation of carefully prepared samples would be highly desirableSeveral methods exist to overcome these limits The simplest method is to reanalyze a sample in multiple targeted assays at the cost of increased time and subject to exhaustion of the sample A new method has been proposed by Domon and colleagues that relies on realtime triggering of targeted assays based on detection of internal standard signals but this method requires that all possible targets are determined prior to analysis have readily available internal standard peptides and may be limited to certain hardware platforms 13Comprehensive mass spectrometry techniques such as dataindependent acquisition DIA and SWATH 14 15 hold great promise for deep interrogation of samples with reproducible detection of analytes across large sample sets However most quantitative applications of these comprehensive techniques have been limited to labelfree modes with attendant difficulties in longitudinal quantitative sample comparison over months or years We sought to take advantage of this mode of data acquisition but with robust quantification for a panel of analytes for which we had previously configured a PRM assay and had obtained SIL internal standard peptides 11 This assay focuses on phosphosignaling and contains a phosphopeptide enrichment step prior to analysis In a perfect world we would run each sample twice once in PRMmode to get the best possible quantification on the selected panel and once in DIAmode to identify and quantify additional analytes not found in the original panel design However this comes at a 2× cost in instrument time and for high throughput labs that analyze large numbers of samples this may not be feasible So we searched for a compromise method where we could experience the benefits of SIL quantification for the selected panel of analytes plus the ability to mine for additional analytes after data analysis while keeping acquisition time the same A hybrid targeted/comprehensive MS assay would allow us to quantify our assay panel while preserving the ability to reinterrogate for new analytes of high value to signaling studies after data acquisitionAs a first step we wanted to ensure that the ratiometric quantification of our analyte panel agreed between the targeted and comprehensive approaches Although in practice it was very easy to acquire data using the comprehensive “DIA” technique with the added benefit of obviating any scheduling requirements robust quantification of our analyte panel proved to be more difficult More specifically the quantitative ratios derived by comparing the signals from endogenous analytes to SIL standards in the DIA assay did not agree with the gold standard fully targeted and scheduled PRM assay when attempting to use identical transitions for quantification Several obvious factors contributed to this disagreement First in many cases both the endogenous and SIL standard are coisolated in the same precursor window Therefore any fragments selected that did not carry a SIL amino acid in the internal standard were effectively unfit for quantification Second the wider precursor isolation windows attendant to the comprehensive technique introduce more possibilities for interference from coeluting analytes

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