Nestle NDC Askeaton - MRM_data

Reduction of MRM protein target list using correlation analysis
Data License: CC BY 4.0 | ProteomeXchange: PXD036543 | doi:
  • Organism: Bos taurus
  • Instrument: 6495A Triple Quadrupole LC/MS
  • SpikeIn: No
  • Keywords: dairy, quantitative proteomics, correlation analysis, MRM, MFGM
  • Lab head: Holger Ebhardt Submitter: Holger Ebhardt
High mass resolution mass spectrometry provides hundreds to thousands of protein identifications per sample, and quantification is typically performed using label-free quantification. However, the gold standard of quantitative proteomics is multiple reaction monitoring (MRM) using triple quadrupole mass spectrometers and stable isotope reference peptides. This raises the question how to reduce a large data set to a small one without losing essential information. Here we present the reduction of such a data set using correlation analysis of bovine dairy ingredients and derived products. We were able to explain the variance in the proteomics data set using only 9 proteins across all major dairy protein classes: caseins, whey, and milk fat globule membrane proteins. We term this method Trinity-MRM. The reproducibility of the protein extraction and Trinity-MRM methods was shown to be below 5% in independent experiments (multi-day single-user and single-day multi-user) using double cream. Further application of this reductionist approach might include screening of large sample cohorts for biologically interesting samples before analysis by high-resolution mass spectrometry or other omics methodologies.
Experiment Description
MRM Sample Preparation (Liquid) Unless otherwise stated, chemicals were purchased from Sigma-Aldrich, a division of Merck. The 18.2 MΩ·cm water was generated on site using a Veolia Purelab Flex 2 purification system. For Trinity-MRM version 1 development, double cream (DC) was purchased from the local grocery store in Ireland and contained 48% fat and 2.1% protein as per label declaration. The DC was kept in a 4°C fridge for storage, and aliquots were taken periodically to perform all subsequent steps at room temperature (20–23°C). The DC sample was weighed 0.2 g ± 10% directly into a microcentrifuge tube using a micro-balance (Mettler Toledo NewClassic MF, model no. MS204S) and values recorded for subsequent data analysis. Subsequently, 0.4 mL of chloroform and 0.4 mL of methanol (both purchased from VWR, a division of Avantor Sciences) were added to the microcentrifuge tube containing DC. Following vigorous vortexing for 30 s, the emulsion was separated by centrifugation at 3,000 × g at 15°C for 5 min. After centrifugation, the protein material was localized between the 2 liquid phases as a white disk (Braakman et al., 2015). Both methanol and chloroform phases were carefully removed using a micropipette, leaving the white disk containing proteins in the microcentrifuge tube. This white disk is resuspended in 0.18 mL of urea 1 M/ABC 50 mM buffer and incubated at 60°C for denaturation for 30 min at 500 rpm using a thermal shaker (Eppendorf ThermoMixer C). For reduction, 5 µL of 1,4-dithiothreitol 0.2 M were added and incubated at 58°C for 15 min (Suttapitugsakul et al., 2017). Both incubations were carried out in a thermal shaker at 500 rpm. Alkylation was carried out by adding 5 µL of iodoacetamide 0.4 M at room temperature (approximately 20–24°C) in the dark for 15 min. Then 10 µL of trypsin 10 µg/µL (Trypsin recombinant, proteomics grade, Roche 03708969001) were added and incubated overnight at 37°C in a thermal shaker at 500 rpm. After overnight incubation, 20 µL of acetonitrile/formic acid (100 mL/2 mL) plus 5 µL of the spike-in peptide mix were added. After briefly vortexing, the samples were centrifuged at 12,000 × g at 4°C for 5 min. After centrifugation, 0.175 mL of the supernatant containing tryptic peptides was transferred into an MS vial, and 5 µL were injected per LC-MS/MS run. MRM QQQ LC-MS/MS Method For details on Trinity-MRM, see Supplemental Table S2 (, which contains the acquisition method report of the Agilent QQQ 6495A mass spectrometer coupled to an Agilent 1290 series HPLC. The C18 reverse-phase columns were Agilent Zorbax analytical column SB-C18 2.1 × 5 mm 1.8 µm (P.N. 821725-902) with pre-column SB-C18 2.1 × 100 mm 1.8 µm (P.N. 858700-902). Stable isotope-labeled peptides were synthesized by Sigma-Aldrich and Thermo Scientific, containing C-terminal [13C6,15N4]Arg or [13C6,15N2]Lys to >95% purity. MRM Data Analysis Result files were analyzed using Skyline software (64-bit, version to determine the area under the curve (total area). A report generated by Skyline was analyzed using Microsoft Excel (Microsoft 365 for Enterprise) and plotted using RStudio (version 1.2.1335), R (version 4.0.2), and pheatmap library (version 1.0.12). Coefficient of variation calculations of repeatability study were carried out as previously described (Bringans et al., 2020).
Sample Description
Dairy Samples Quantified All dairy samples are of bovine origin (Taxonomy 9913 Bos taurus). Commercially available whey protein concentrate (WPC) products of different protein and fat contents were sourced from the EU, USA, and New Zealand, prepared in all cases from sweet whey: 5 batches of a high-fat WPC enriched at 30% α-lactalbumin (WPC_alac), 3 batches of whey protein enriched at 41% α-lactalbumin, and 2 regular WPC containing 70% and 35% protein, respectively. Because many new MFGM products were available on the market, 4 different products were sourced with up to 5 batches per product (MFGM_P1 through MFGM_P4). For comparative purposes, 3 batches of skim milk powder (SMP) were added.
Created on 9/5/22, 2:50 PM
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