KTH Uhlen Lab - Multiple Proteases

Addressing the Protease Bias in Quantitative Proteomics
Data License: CC BY 4.0 | ProteomeXchange: PXD033574 | doi: https://doi.org/10.6069/6pyn-aw16
  • Organism: Homo sapiens
  • Instrument: TSQ Altis
  • SpikeIn: Yes
  • Keywords: Multiple proteases, SRM, Targeted proteomics, Absolute quantification, Plasma proteomics
  • Lab head: Fredrik Edfors Submitter: Jakob Woessmann
Protein quantification strategies using multiple proteases have been shown to deliver poor inter-protease accuracy in label-free mass spectrometry experiments. By utilizing six different proteases with different cleavage sites, this study explores the protease bias and its effect on accuracy and precision by using recombinant protein standards. We established 557 SRM assays, using a recombinant protein standard resource, towards ten proteins in human plasma and determined their concentration with multiple proteases. The quantified peptides of these plasma proteins spanned three orders of magnitude (0.02-70 µM). In total, 60 peptides were used for absolute quantification and the majority of the peptides showed high robustness. The retained reproducibility was achieved by quantifying plasma proteins using spiked stable isotope standard recombinant proteins in a targeted proteomics workflow.
Experiment Description
To establish SRM assays, protein epitope signature tags (PrESTs, up to 149 amino acids long) were digested individually by six different proteases. PrESTs were reduced by Dithiothreitol (DTT, 10 mM, 30 min, 56 °C) followed by alkylation with 2-Chloroacetamide (CAA, 50 mM, 30 min, Room Temperature (RT)) in the dark. For the digestion with Lysarginase (Sigma-Aldrich), Endoproteinase Arg-C (Roche) and Chymotrypsin (Thermo Scientific) CaCl2 was added to a final concentration of 10 mM. The volume of the Trypsin (Thermo Scientific), GluC (Thermo Scientific) and LysC (Wako) digestion was adjusted respectively by 1x PBS. Digestion was performed in a 1:20 Enzyme to Protein ratio (E:P) overnight and was quenched by addition of TFA to a final concentration of 0.5 %. HPLC vials (Waters) were equilibrated in the auto sampler for 1h prior to analysis. During the method development, two pmol PrEST peptides were injected. Plasma samples of three healthy males and two females were pooled. For the absolute quantification of plasma proteins the plasma was spiked with SIS PrESTs. SIS PrEST concentration was adjusted to endogenous levels. The Plasma- SIS PrEST mixture was diluted 30x with 1 % sodium deoxycholate (SDC) and 1 M Urea. Samples were reduced at a final DDT concentration of 10 mM (30 min, 37 °C) and alkylated with CAA (50 mM, 30 min, RT in the dark). The Plasma SIS PrEST Pool was split into 18 vials holding 1 µL plasma each. SDC was diluted to 0.1 % with 1x PBS and for the digestion with Lysarginase, ArgC and Chymotrypsin CaCl2 was added to a final concentration of 10 mM. The volume of the Trypsin, GluC and LysC digestion was adjusted respectively by 1x PBS. Digestion with ArgC (1:50 E:P), Chymotrypsin (1:50 E:P), GluC (1:50 E:P), LysC (1:50 E:P), Lysarginase (1:20 E:P) and Trypsin (1:50 E:P) were each performed in triplicate. Digestion was performed overnight and was quenched by addition of TFA to a final concentration of 0.5 %. Approximately 50 µg of plasma digest was desalted by means of 6 layer C18 (Empore Octadecyl C18 47mm Extraction Disks 2215, CDS Analytical) StageTips prepared in-house. The C18 membrane was activated by 100% ACN and equilibrated by 0.1 % TFA. Plasma SIS PrEST digest was added and the stage tips were washed two times with TFA 0.1 %. The samples were eluted with 80 % ACN, 0.1 % FA. Stage tips were centrifuged after each step at 931 x g for 3 min. The eluate was vacuum dried at 45 °C and stored at -20 °C until analysis. For LC-MS/MS analysis, the samples were resuspended in Solvent A. Vials were equilibrated in the autosampler for 1h before analysis. 12 µg plasma SIS PrEST digest was injected in triplicates in random order.
Sample Description
Human plasma pool from five healthy individuals (3 male, 2 female) with spiked standards.
Created on 5/3/22, 10:35 PM
Clustergrammer Heatmap
220124_PrEST_Assays_ArgC_select_top10_2022-05-03_14-29-07.sky.zip2022-05-03 22:41:0210383837520
220124_PrEST_Assays_Trypsin_select_top10_2022-05-03_14-27-38.sky.zip2022-05-03 22:40:58111451451,34920
220124_PrEST_Assays_GluC_select_top10_2022-05-03_14-02-14.sky.zip2022-05-03 22:40:381110510598620
220124_PrEST_Assays_Chymotrypsin_select_top10_2022-05-03_13-12-26.sky.zip2022-05-03 22:40:3311979790920
220124_PrEST_Assays_Lysarginase_select_top10_2022-05-03_12-52-37.sky.zip2022-05-03 22:40:28111291291,22720
220124_PrEST_Assays_LysC_select_top10_2022-05-03_10-40-54.sky.zip2022-05-03 22:40:2311777771020
211119_AllTargets_Trypsin_select_top10_quantification_cleaned_top5_2022-01-31_14-43-11.sky.zip2022-05-03 22:40:19526522549
211103_AllTargets_LysC_select_top10_quantification_cleaned_top5_2022-01-31_14-37-29.sky.zip2022-05-03 22:38:48411221109
211108_AllTargets_Lysarginase_select_top10_quantification_Cleaned_top5_2022-01-31_14-33-00.sky.zip2022-05-03 22:38:20318361809
211123_AllTargets_GluC_select_top10_quantification_cleaned_top5_2022-01-31_14-25-37.sky.zip2022-05-03 22:37:494612549
211123_AllTargets_Chymotrypsin_select_top10_quantification_cleaned_top5_2022-01-31_14-21-55.sky.zip2022-05-03 22:34:584816709
211108_AllTargets_ArgC_select_top10_quantification_cleaned_top5_2022-01-31_14-11-04.sky.zip2022-05-03 22:34:58324209