DOI: 10.5061/dryad.1rn8pk0xm
title: Serum proteomic analysis of sex differences during an acute low back pain episode

description: Growing evidence suggests that pain and injury engage biological processes
 and systems that extend beyond the nervous system. The molecular processes
 driving the transition from acute to chronic low back pain (LBP) remain
 poorly understood. Here, we explore the serum proteomic profile of male
 and female participants during an acute LBP episode (N=59). Differential
 protein expression during the acute-stage of LBP was compared between
 participants with resolved LBP at three months and those with chronic or
 recurrent LBP at three-months. Proteome-wide analysis using
 mass-spectrometry identified 216 proteins confidently. Sex differences in
 protein abundance changes were evident upon inspection of fold changes.
 Multivariable data analysis identified 21 serum proteins during the acute
 episode that correctly classified 93% of males and 23 serum proteins that
 correctly classified 90% of females with ongoing LBP at three months. Most
 of the differentially expressed proteins during acute LBP were involved in
 immune, inflammatory, complement or coagulation responses. Overall, males
 who recovered from an acute LBP episode had a greater tendency to
 demonstrate an upregulated immune-driven inflammatory response during
 acute LBP, whilst women with pain resolution demonstrated a tendency
 towards a downregulated immune response. Taken together, this study
 provides data to suggest biological processes during an acute LBP episode
 may contribute to resolution, or persistence, of LBP symptoms at three
 months, however, these processes differ between sexes. This work provides
 an early foundation for future research exploring strategies targeting
 distinct immune system processes in men and women that may interfere with
 the transition from acute to chronic LBP. 
description: Sample
 preparation Peripheral venous blood was
 drawn into serum tubes (BD, SST II Advance) through venepuncture of the
 median cubital vein at baseline assessment. The sample was clotted (30
 min, room temperature) and then separated by centrifugation (2500 rpm, 15
 min). Samples were pipetted into 50 μL aliquots and immediately stored at
 -80°C. After thawing, de-identified serums from all 59 participants were
 prepared by digesting 3µl of serum (57µg ul<sup>-1</sup>
 +/-7µg) in 50µl of 50mM AMBIC, 2M urea, 10mM DTT at pH 8 using trypsin at
 25°C for 16 hours in a 1:100 enzyme to protein ratio. Digestion was halted
 by acidification. Serum peptides were fractionated using hydrophobic
 interaction chromatography (HILIC) according to the manufacturer's
 protocol (PolyLC Inc, MD, USA) with the additional parameter of decreasing
 solvent-releasing increasingly hydrophilic peptides. Five fractions with
 decreasing solvent were prepared (unbound fraction, 25%,50%,70%, and 100%
 sequential solvent extraction) with peptides released by 70% acetonitrile
 in 15mM ammonium acetate fraction being evaluated further.
 Mass spectrometry of
 samples Digested and fractionated peptides were
 reconstituted in 5μL 0.1% formic acid and separated by nano-LC using an
 Ultimate 3000 HPLC and autosampler (Dionex, Amsterdam, Netherlands). The
 sample, (0.6μL from 5μl), was loaded onto a micro C18 pre-column (300
 μm×5mm, Dionex) with
 H<sub>2</sub>O:CH<sub>3</sub>CN (98:2, 0.1% TFA)
 at 10μL min<sup>−1</sup>. After washing, the pre-column was
 switched (Valco 10 port valve, Dionex) into line with a fritless
 nanocolumn (75μm id×12 cm) containing reverse phase C18 media (1.9μm, 120
 ̊A, Dr. Maisch GmbH HPLC). Peptides were eluted using a linear gradient of
 H<sub>2</sub>O:CH<sub>3</sub>CN (98:2, 0.1% formic
 acid) to H<sub>2</sub>O:CH<sub>3</sub>CN (64:36,
 0.1% formic acid) at 250nlmin<sup>−1 </sup>over 90 min. The
 QExactive (Thermo Electron, Bremen,Germany) mass spectrometer (MS) was run
 in DDA mode as previously described (58). Protein identification relative
 quantitation Protein dataset-peak lists were generated from
 raw files using Mascot Daemon v2.5.1 (Matrix Science, London, UK,
 www.matrixscience.com). All MS/MS spectra were searched against Swissprot
 (downloaded February 2018; 556,568 sequences for protein identification
 with the following criteria: 1) species, Human; 2) allowed 1 missed
 cleavage; 3) variable modifications, Oxidation (M), phosphorylation (S, T,
 Y); 4) peptide tolerance, ±5 ppm; 5) Fragment tolerance, ±0.05 Da; 6)
 peptide charge+2 and +3; and 7) enzyme specificity, semi-tryptic. A decoy
 database search was also performed. Only proteins identified from the
 Swissprot database, controlled by the Benjamini-Hochberg procedure for
 multiple comparisons, with two or more unique peptides were included in
 further analysis. 
description: ThermoScientific Xcalibur software for *.raw
 files Microsoft XLS
description: # Serum proteomic analysis of sex differences during an acute low back
 pain episode This study
 used a sub-sample of participants from the Understanding persistent Pain
 Where it ResiDes (UPWaRD) longitudinal cohort who provided a serum sample
 at baseline assessment (ACTRN12619000002189) (1). This study included 59
 participants matched in age and sex (30 females [mean age 42 ± 15]; 29
 males [mean age 38±16]) who experienced an acute LBP episode. Further, an
 equal number of participants from the UPWaRD cohort who were recovered or
 not-recovered at three months were included. Specifically, 15 male and 15
 female participants were considered recovered from their LBP episode (NRS
 = 0.30 ± 0.47), while 14 male and 15 female participants were considered
 not recovered (NRS = 4.0 ± 1.7) at three-month follow-up. Scaffold
 Software (version 4.8.7, Proteome Software Inc., Portland, OR) was used to
 compare the shotgun proteomic results using spectral counting. Peptide
 identifications were accepted if they could be established at greater than
 95% probability using the Scaffold delta-mass correction. Expression
 changes across samples were measured via spectral count, normalised by
 total ion count with missing values kept at zero and recorded as not
 detected (ND) during further analysis. The deposited data consists of the
 raw mass spectrometry files and the xls spreadsheet output using Scaffold
 software. 1.     Jenkins LC, Chang W-J, Buscemi V, Liston M, Nicholas M,
 Graven-Nielsen T, et al. The Understanding persistent Pain Where it
 ResiDes study of low back pain cohort profile. medRxiv.
 2021:2021.11.24.21266794.