David L. Tabb, Ph.D. January 11, 2017 Broadening the mission: quality assessment for quantitative and label-dependent mass spectrometry David L. Tabb, Ph.D. January 11, 2017

Organization Introducing the HUPO-PSI QC Working Group Assessing isobaric labeling: Spectrum Mill Understanding U.S. Pharmacopeia Chapter 621 Assessing quality in targeted expts: SProCoP/MSstats Extending into MSE and SWATH Planning for MALDI profiling and imaging

HUPO-PSI QC Working Group Weimin Zhu Stefan Tenzer Wout Bittremieux Dave Tabb Mathias Walzer (plus not pictured) Martin Eisenacher Reza Salek Produce format, CV terms, and infrastructure to analyze, visualize, and convey quality information http://www.psidev.info/groups/quality-control

QC infrastructure serves many purposes Figure by Wout Bittremieux

Spectrum Mill innovations Tag-based searching Ion series-based scoring Raw data preprocessing Homology searching AA substitution Wildcard PTMs QC metric producing Per-MS/MS assessing http://proteomics.broadinstitute.org/

iTRAQ labeling After digestion, amino groups of peptides can be attached to isobaric tagging reagents. Tags fragment in MS/MS to produce reporter ions, depending on tag type. AB Sciex sells 4- and 8-channel reagents. Ross et al (2004) Mol. Cell. Proteomics 3: 1154-1169.

Why do isobaric tags introduce variability? iTRAQ reagents bond to primary amines both at N-terminus and on lysine. Lab errors may alter label rxn efficiency. Fold changes are biased toward 1:1 ratios. D.W. Mahoney et al. J. Proteome Res. (2011) 10: 4325-4333

Spectrum Mill 6.0 isobaric QC For what fraction of MS/MS scans is label observed at N-term? At Lys? Neither? Both? N-term is typically less than complete What is the ratio of intensity for signal at a given reporter to most intense (across all MS/MS)? What was median signal/noise ratio for reporter ion intensities? Insights from Karl Clauser, Broad Institute

USP*: Useful Starting Point *Actually Pharmacopeia of the United States of America, 38th Rev.

USP Chapter 621 on Chromatography defines: Resolution Separation Factor Symmetry Factor Signal-to-Noise Ratio %RSD Requirements

Tools for SRM/XIC evaluation SProCoP (Statistical Process Control in Proteomics): Bereman et al. JASMS (2014) 25: 581-587. MSstats: Choi et al. Bioinformatics (2014) 30: 2524-2526.

SProCoP: key QC visualizations Shewhart Process Control Chart Pareto Chart Variability contribution +2SD +1SD Mean -1SD -2SD Intervention Bereman et al. JASMS (2014) 25: 581-587

MSstats v3.7.1 differentiates, finds LOD, and monitors QC DDA SRM DIA MSstats can accommodate intensity data from any of these experiment designs, using peptide XICs (DDA) or peptide fragment XICs (SRM,DIA). Visualization of one representative protein in a DDA, an SRM and a DIA experiment. Colors and shapes represent peptides, and multiple line types of a same color and shape represent the fragments of the peptide. Vertical lines separate times or conditions. (a) Protein alcohol dehydrogenase-yeast spiked into a complex background in six concentrations. (b) Protein ACH1, at 10 times points after a stress. (c) Protein FabG of Streptococcus, with 0 and 10% human plasma added http://msstats.org/ Choi et al. Bioinformatics (2014) 30: 2524-2526.

Specializing QC metrics to DIA Is cycle time a function of TIC? Dot products can compare successive scans for a swath. Mass precision can be examined through repeat scans. Gillet et al. Mol. Cell. Proteomics (2012) 10.1074/mcp.O111.016717–3

Opportunities in DIA precursor-fragment correlation What fraction of MS isotope packets match fragments? What is median RT diff for peak maxima? How close to an exponential-Gaussian is elution profile? Retention Time Geromanos et al. Proteomics (2009) 9:1683 Broeckling et al. Metabolomics (2012) 9:33 Kalambet et al. Chemometrics (2011) 25: 352

QC for MALDI Profiling Within-cohort variability can be assessed on both m/z and intensity. Matrix-associated peaks may reveal spot- dependent effects. m/z mapping benefits from mass accuracy more than resolution.

QC for MALDI Imaging Spatial coords imply intensity similarity. Is intensity “noise” independent of m/z? TIC histogram may highlight regions of poor signal.

Closing Thoughts Proteomic paradigms beyond DDA and SRM need more support in QC toolsets. Sometimes essential work can appear less glorious, at first glance. That does not make it nonessential. The HUPO-PSI QC Working Group is looking for brains and hands!