![]() Mascot, Andromeda, Spectrum Mill, MSGF+) and a RefProtDB FASTA file with Entrez GeneID information for inference ( supplemental Fig. GpGrouper uses peptide spectral matches (PSMs) data produced by a search engine of user's choice ( e.g. For the WHIM PDX profiling samples, an older original sRP protocol ( This is our current standard sRP configuration that allows identification and quantification of ∼6000–7000 proteins per 5 μg of peptide in ≤12 h. For label-free human and mouse mixture profiling samples, a total of 15 (2–30% ACN, 2% steps) fractions were obtained and combined into 5 pools for mass spectrometry sequencing (15F5R protocol with 02 + 12 + 22, 04 + 14 + 24, 06 + 16 + 26, 08 + 18 + 28, and 10 + 20 + 30% combinations). This is the most extensive sRP protocol used here to achieve better separation of peptide complexity and minimize ratio compression in isobaric methods. ![]() ![]() For TMT-tagged human and mouse peptide mixtures, a total of 18 fractions (2–36% ACN, 2% steps) were collected and combined into 6 pools (18F6R protocol with 02 + 14 + 26, 04 + 16 + 28, 06 + 18 + 30, 08 + 20 + 32, 10 + 22 + 34, and 12 + 24 + 36% combinations). Bound peptides were fractionated by elution with a step gradient of 150 μl of increasing concentrations of ACN, combined into pooled fractions non-contiguously, and vacuum dried. Vacuum-dried peptides were dissolved with 150 μl of pH10 ABC buffer and loaded on the C18 tip pre-equilibrated with same pH10 ABC solution. Maisch GmbH, Germany) on top of a C18 disk plug (Empore TM C18, 3M). A micropipette tip with a C18 resin was made from a 200 μl pipette tip by layering 6 mg of C18 matrix (Reprosil-Pur Basic C18, 3 μm, Dr. Vacuum-dried peptides were dissolved in pH10 ABC buffer (10 m m ammonium bicarbonate, pH 10, adjusted by NH 4OH) and subjected to off-line microscaled reverse phase separation (or “sRP”, for small scale RP). Off-Line Basic pH Reverse Phase Peptide Fractionation After labeling peptides for 1 h at room temperature, reactions were quenched by adding 8 μl of 5% hydroxylamine solution and incubating for another 15 min. 0.8 mg vial of TMT Label Reagent TMT-126 or 129N was dissolved in 41 μl anhydrous acetonitrile, added to 50 μg peptides in 100 μl 50 m m TEAB of HeLa or NIH-3T3, respectively. After digestion, peptide concentration was measured using Pierce Quantitative Colorimetric Peptide Assay (Catalogue #23275, Thermo Fisher Scientific). Dried protein was dissolved in 100 μl 50 m m TEAB and digested with 25 ng trypsin per 1 μg protein overnight at 37 ☌. The protein was pelleted by centrifugation and air dried for 3 min. Total protein was precipitated with 6-volumes of cold acetone overnight at −20 ☌. Five microliters of 200 m m TCEP was added to each sample for 1 h at 55 ☌, then 5 μl of 375 m m iodoacetamide was added to sample for 30 min at room temperature in the dark. Fifty micrograms of protein per sample was transferred to a new tube and adjusted to a final volume of 100 μl with 100 m m TEAB. The protein concentration of the supernatant was measured using Bradford Assay (Catalogue #23238, Thermo Fisher Scientific). The lysate was then cleared by centrifugation at 21,000 rcf for 20 min at 4 ☌. The lysate was sonicated for 10 s at 20% power for 3 times with interval 30 s on ice (Ultrasonic Processor VC 505, Sonics & Materials BE). Briefly, HeLa and NIH-3T3 cell pellets were lysed in Lysis Buffer (100 m m TEAB with 1% SDS). ![]() HeLa or NIH-3T3 peptides were labeled with isobaric tandem mass tags (TMTsixplex, Catalogue #90061, Thermo Fisher Scientific) according to the manufacturer's instructions. 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. This is a critical capability for proper evaluation of proteomics data from PDX samples, where stromal infiltration varies across individual tumors. Furthermore, gpGrouper seamlessly handles two-species samples such as patient-derived xenografts (PDXs) without ignoring the host species or species-shared peptides. We experimentally show that distributing shared peptide quantities based on unique peptide peak ratios improves quantitation accuracy compared with conventional winner-take-all scenarios. 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. In quantitative mass spectrometry, the method by which peptides are grouped into proteins can have dramatic effects on downstream analyses. ![]()
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