TITLE: MRM-LC-MS/MS Assay for inflammatrory associated proteins in serum
AUTHORS:  

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Multiplex targeted proteomic assay to measure human proteins associated with inflammation in serum. A unique peptide is selected to represent a protein and is measured using multiple reaction monitoring (MRM) mass spectrometry. </div></div>

[STEPS]
?. [Sample preparation]
samples are  prepared for targeted mass spectrometric analysis utilising 20 μL serum.
?. [Sample preparation]
Prior to preparation, 5 pmol yeast enolase 1 protein (ENO1) (sigma UK) were added to each 20 μL serum sample to act as an internal standard, accounting for variation in sample preparation and instrumental analysis.
?. [Sample preparation]
To reduce the complexity of the sample matrix and enable the detection of low- and medium abundant proteins, the serum was precipitated with 40 μL 3M ammonium sulphate.
?. [Sample preparation]
This was followed by incubation at room temperature for 10 minutes before centrifugation at 16900 x g for 10 minutes to separate supernatant from protein pellet.
?. [Sample preparation]
The supernatant was discarded and the protein pellet was washed with 50 μL 1.8M ammonium sulphate.
?. [Sample preparation]
The proteins were thereafter solubilised in 20 μL digestion buffer containing 6M urea, 2M thiourea, 2% ASB-14 and 100mM Tris, pH7.8, and shaken until all protein pellet had been re-suspended.
?. [Sample preparation]
To reduce sulphide bonds, 45 μg dithioerythriol was added and the samples were shaken for 60 minutes.
?. [Sample preparation]
To prevent the sulphide bonds from reforming, 108 μg iodoacetic acid were added and the samples were shaken for another 60 minutes.
?. [Sample preparation]
165 μl  MilliQ water was added to dilute the concentration of urea
?. [Sample preparation]
add 1 μg trypsin (Promega, UK) was added to digest the proteins into tryptic peptides.
?. [Sample preparation]
The samples were incubated in a +37 °C water bath for 16 hours.
?. [Sample preparation]
In order to remove salts, a solid phase extraction (SPE) was performed on a 100 mg Bond Elut C18 96 well plate (Agilent, Santa Clara, CA, United States). Prior to SPE clean up, the samples were adjusted to a concentration of 0.1% trifluoroacetic acid (TFA).
?. [Sample preparation]
The SPE beds were washed with 1 mL 60% acetonitrile (ACN), 0.1% TFA before equilibration by two 1 mL aliquots of 0.1% TFA.
?. [Sample preparation]
The samples were loaded onto the beds and salts were washed away by the addition of 1 mL 0.1% TFA before elution of the desalinated samples by two 250 μL aliquots of 60% ACN, 0.1% TFA.
?. [Sample preparation]
Solvents were evaporated using a SpeedVac (Eppendorf, Hamburg, Germany).
?. [LC-MS/MS Analysis]
Before mass spectrometric analysis, the samples were reconstituted in 50 μL 3% ACN, 0.1% TFA.
?. [LC-MS/MS Analysis]
Utilising a Waters Acquity Ultra Performance Liquid Chromatography system (Waters, Manchester, UK), 4 μL of sample was injected onto a Waters 50 mm CORTECS® UPLC® C18 1.6 µm column fitted with a 5 mm VanGuard column of the same chemistry, operating at 45 °C, for chromatographic separation. The mobile phase consisted of A: 0.1% formic acid in water and B: 0.1% formic acid in ACN, pumped at a flow rate of 0.6 mL min-1. The starting conditions of 3% B were kept static for 0.1 minutes, before initialising the linear gradient utilised to elute and separate the peptides over 7.7 minutes to 40% B. B was thereafter linearly increased to 80% over 0.2 minutes and held for 1 minute to wash the column before returning to the initial conditions followed by equilibration for 1 minute prior to the subsequent injection. The LC system was coupled to a Waters Xevo-TQ-S triple quadrupole mass spectrometer for multiple reaction monitoring (MRM) detection in positive electrospray ionisation mode. The capillary voltage was set to 2.8 kV, the source temperature to 150 °C, the desolvation temperature to 600 °C, the cone gas and desolvation gas flows to 150 and 1000 L hour-1 respectively. The collision gas consisted of nitrogen and was set to 0.15 mL min-1. The nebuliser operated at 7 bar. The cone energy was set to 35 V and the collision energies varied depending on the optimal settings for each peptide. Three injections were performed for each sample.
?. [Data Processing]
After acquisition, the data were imported to Skyline open source software (https://skyline.ms/project/home/software/Skyline/begin.view). The quantifier and qualifier ions were inspected and compared to a pooled sample spiked with increasing amounts of synthetic peptides to ensure that the correct peaks were identified and integrated. The relationships between quantifier and qualifier ions were evaluated for consistency across the samples and the most abundant peptide ions were chosen for quantitation. The integrated areas were exported to Microsoft Excel and the analyte quantifier areas were normalised to yeast enolase 1 to render ratios. The relative response of each analyte to the response of the known concentration of the internal standard was used to estimate the concentration of the proteins in the samples.
?. To evaluate the stability of the assay, five repeated injections of 1 pmol synthetic standards were performed and the coefficients of variation were calculated. The coefficients of variation for the most significant compounds were 14% for protein NDRG1 (NDRG1), 20% for carbamoyl-phosphate synthase [ammonia], mitochondrial (CPSM), 9% for cystatin C (CST3), 3% for progranulin (GRN),  5% for collagen triple helix repeat-containing protein 1 (CTHRC1)	and 4% for thioredoxin-dependent peroxide reductase, mitochondrial (PRDX3).
?. List of proteins and corresponding peptides included in the multiplex inflammation panel assay