Searle_PREGO_manuscript

Searle_PREGO_manuscript
Analytical protein standards
  • Organism: Homo sapiens
  • Instrument: TSQ Vantage
  • SpikeIn: No
Abstract
Targeted mass spectrometry is an essential tool for detecting quantitative changes in low abundant proteins throughout the proteome. Although Selected Reaction Monitoring (SRM) is the preferred method for quantifying peptides in complex samples, the process of designing SRM assays is laborious. Peptides have widely varying signal responses dictated by sequence-specific physiochemical properties; one major challenge is in selecting representative peptides to target as a proxy for protein abundance. Here we present PREGO, a software tool that predicts high responding peptides for SRM experiments. PREGO predicts peptide responses with an artificial neural network trained using 11 minimally redundant, maximally relevant properties. Crucial to its success, PREGO is trained using fragment ion intensities of equimolar synthetic peptides extracted from data independent acquisition (DIA) experiments. Due to similarities in instrumentation and the nature of data collection, relative peptide responses from DIA experiments are a suitable substitute for SRM experiments because they both make quantitative measurements from integrated fragment ion chromatograms. Using an SRM experiment containing 12973 peptides from 724 synthetic proteins, PREGO exhibits a 40-85% improvement over previously published approaches at selecting high responding peptides. These results also represent a dramatic improvement over the rules-based peptide selection approaches commonly used in the literature.
Experiment Description
An SRM training validation data set was constructed using the protocols presented in Stergachis et al. Briefly, clones for GST fusion proteins from the pANT7_cGST clone collection (https://dnasu.org/DNASU/Home.do) were synthesized in vitro using the Pierce 1-step Human Coupled in vitro protein synthesis kit (Thermo Scientific; Bremen, Germany). In instances where a cDNA clone was unavailable, recombinant proteins were simply purchased from a commercial source. GST tagged proteins were captured using glutathione sepharose 4B beads (GE Healthcare Life Sciences; Pittsburgh, PA), and iteratively washed to remove nonspecific binders. Bead bound GST fusion proteins were individually denatured with 5 mM dithiothreitol (DTT) for 30 minutes at 60 °C and alkylated with 15 mM iodoacetic acid (IAA) for 30 minutes at room temperature. Proteins were then digested 1 ug of sequencing grade modified porcine trypsin (Promega; Madison, WI) for 2 hours at 37°C.
Created on 4/30/15, 11:44 AM

Library Statistics:

Clustergrammer Heatmap
 
Download
Human_Prothrombin_P00734_Open_Edit_with_iRT_121213_2015-01-30_17-04-21.sky.zip2015-04-30 12:28:32125252051
LCAT_Template_2015-01-30_15-11-22.sky.zip2015-04-30 12:26:26188631
Human_Factor_IX_P00740_with_iRT_092413A_Library_2015-01-30_15-08-31.sky.zip2015-04-30 12:24:3611212891
Human_Factor_X_P00742_with_iRT_092413A_Library_2015-01-30_15-07-10.sky.zip2015-04-30 12:22:34113131031
Human_Complement_4_P0C04A_Open_120513_Library_2014-12-10_12-02-42.sky.zip2015-04-30 12:19:23148483212
Human_Fibronectin_P02751_Open_Library_edit_08_19_2014_2014-12-08_13-43-11.sky.zip2015-04-30 12:03:17154543831
HGA1 and HGG1_library_2014-12-05_17-06-43.sky.zip2015-04-30 12:01:45288701
Chromatogram_Library_08_19_2014_Build_DBS_Targets_2014-09-02_14-27-37.sky.zip2015-04-30 11:58:20541,0321,0327,37453
Human_ORM1_P02763_Open_Library_edit_08_19_2014_2014-08-22_16-21-09.sky.zip2015-04-30 11:55:5211010761
Human_APOB100_P04114_Open_Edit_with_iRT_120913_Figures_2014-06-09_11-19-51.sky.zip2015-04-30 11:54:0722022027602
Human_Angiotensinogen_P01019_Open_Library_Edit_121613_2014-02-18_12-25-38.sky.zip2015-04-30 11:49:31114141271
Human_GPT1_P24298_Wide_Open_120213_2013-12-06_15-59-13.zip2015-04-30 11:48:1711313901
Human_ALDOA_P04075_Open_112713_2013-12-06_15-53-56.zip2015-04-30 11:47:15117171181
Human_A2M_P01023_Open_120513_2013-12-06_15-51-36.zip2015-04-30 11:45:29163635271