gpGrouper: A Peptide Grouping Algorithm for Gene-Centric Inference and Quantitation of Bottom-Up Proteomics Data

Abstract

In quantitative mass spectrometry, the method by which peptides are grouped into proteins can have dramatic effects on downstream analyses. Here we describe gpGrouper, an inference and quantitation algorithm that offers an alternative method for assignment of protein groups by gene locus and improves pseudo-absolute iBAQ quantitation by weighted distribution of shared peptide areas. We experimentally show that distributing shared peptide quantities based on unique peptide peak ratios improves quantitation accuracy compared with conventional winner-take-all scenarios. Furthermore, gpGrouper seamlessly handles two-species samples such as patient-derived xenografts (PDXs) without ignoring the host species or species-shared peptides. This is a critical capability for proper evaluation of proteomics data from PDX samples, where stromal infiltration varies across individual tumors. Finally, gpGrouper calculates peptide peak area (MS1) based expression estimates from multiplexed isobaric data, producing iBAQ results that are directly comparable across label-free, isotopic, and isobaric proteomics approaches.

Streamlined Protocol for Deep Proteomic Profiling of FAC-sorted Cells and Its Application to Freshly Isolated Murine Immune Cells*

“…The low input proteomics described here extend the applicability of previous methodologies to FAC-sorted immune cells and highlight the value of reduced-handling sample preparation, especially those requiring analysis of limiting amounts of sample (22,23,36). Other applications of the methods we describe include profiling of other cell types, co-immunoprecipitation studies, and post-enrichment labeling.…”

“…TMT10 reporter ion intensities in each MS/MS spectrum were corrected for isotopic impurities by the Spectrum Mill protein/peptide summary module using the afRICA correction method which implements determinant calculations according to Cramer's Rule (50) and general correction factors obtained from the reagent manufacturer's certificate of analysis (https://www.thermofisher.com/order/catalogue/ product/90406) Fractional intensities (frINT) were calculated as the sum of precursor ion chromatographic peak areas in MS1 spectra for all PSMs contributing to the protein. A similar approach has been previously reported (36). frINT for each protein was calculated by splitting the combined precursor ion abundance in proportion to its individual normalized protein-level reporter ion ratio.…”

Partial loss of CFIm25 causes aberrant alternative polyadenylation and learning deficits

“…We processed, measured, and analyzed the sample as previously described (Saltzman et al, 2018). We summarize the main steps below.…”

“…A “15F5R” protocol was used for off-line fractionation as described before(Saltzman et al, 2018). We filled a 200 μl pipette tip with 6 mg of C18 matrix (Reprosil-Pur Basic C18, 3 μm, Dr. Maisch GmbH) on top of a C18 disk plug (EmporeTM C18, 3M).…”

MiR-146a wild-type 3’ sequence identity is dispensable for proper innate immune function in vivo

“…Mass spectrometric analysis was carried out on an Orbitrap Fusion LumosETD (Thermo Fisher) by the Mass Spectrometry Proteomics core at Baylor College of Medicine as described before (Saltzman et al 2018). Briefly, BMDM lysates from 8-12-week old mice were denatured in ABC solution (50 mM ammonium bicarbonate + 1mM CaCl2), with subsequent snap freeze/42 °C thaw cycling and boil denaturing at 95 °C.…”