Serum proteomic analysis of sex differences during an acute low back pain episode
Data files
Dec 18, 2023 version files 50.77 GB
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AD_15.raw
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AJS_17.raw
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AMM_18.raw
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APT_17.raw
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AR_15.raw
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AST_15.raw
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BAT_18.raw
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BAW_15.raw
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BFF_19.raw
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BHM_18.raw
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BTF_18.raw
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CBT_15.raw
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CCM_18.raw
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CCM_19.raw
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CCT_18.raw
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CDM_17.raw
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DBM_15.raw
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DTT_18.raw
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DUF_17.raw
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FSF_16.raw
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FST_17.raw
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GCW_18.raw
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GPT_16.raw
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GST_16.raw
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GTT_17.raw
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HDM_16.raw
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JCW_16.raw
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JHW_15.raw
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JRF_16.raw
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JSM_16.raw
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JZT_16.raw
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KHM_18.raw
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KLT_17.raw
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LFW_16.raw
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LKW_18.raw
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LRW_17.raw
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LSM_17.raw
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MCT_16.raw
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MCW_18.raw
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MGT_16.raw
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MPM_16.raw
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MPT_17.raw
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MST_15.raw
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MWT_15.raw
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NST_18.raw
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OST_16.raw
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POT_16.raw
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PPM_16.raw
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RCW_16.raw
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README.md
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RECOVERED_VS_NON_RECOVERED_MULTIPLE_CORRECTION_01-12-20.xls
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RRT_17.raw
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SBM_18.raw
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SBT_15.raw
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SCF_17.raw
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SGT_17.raw
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SIF_16.raw
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TAF_16.raw
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TMT_18.raw
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VRF_16.raw
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VST_18.raw
Abstract
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.
README: Serum proteomic analysis of sex differences during an acute low back pain episode
https://doi.org/10.5061/dryad.1rn8pk0xm
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.
Methods
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-1 +/-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 H2O:CH3CN (98:2, 0.1% TFA) at 10μL min−1. 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 H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250nlmin−1 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.
Usage notes
- ThermoScientific Xcalibur software for *.raw files
- Microsoft XLS