Serum proteomic analysis of sex differences during an acute low back pain episode

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..., 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 p...,
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, # 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...

越来越多的证据表明，疼痛与损伤所涉及的生物学过程与系统已超出神经系统范畴。目前，驱动急性腰痛（low back pain, LBP）向慢性腰痛（LBP）转化的分子机制仍未得到充分阐明。本研究探究了急性腰痛发作期间男性与女性受试者的血清蛋白质组学特征（N=59）。我们将3个月时腰痛缓解的受试者与3个月时仍存在慢性或复发性腰痛的受试者，在急性腰痛阶段的差异蛋白表达情况进行了对比。通过质谱（mass spectrometry）开展的全蛋白质组分析可靠鉴定出216种蛋白质。对蛋白丰度变化倍数的分析显示，不同性别间存在显著蛋白差异。多变量数据分析显示，在急性发作期，有21种血清蛋白可准确分类93%的男性受试者（3个月时仍存在持续性腰痛），另有23种血清蛋白可准确分类90%的女性持续性腰痛受试者。急性腰痛阶段的差异表达蛋白大多为……

样本制备
在基线评估时，通过肘正中静脉穿刺采集外周静脉血至血清采血管（碧迪，BD SST II Advance）。样本于室温下静置凝血30分钟，随后以2500转每分钟离心15分钟分离血清。将血清分装为50 μL等分试样，并立即置于-80℃冰箱保存。解冻后，对所有59名受试者的去标识化血清样本进行前处理：取3 μL血清（浓度为57 μg·μL⁻¹ ±7 μg·μL⁻¹），在pH 8的50 mM碳酸氢铵（ammonium bicarbonate, AMBIC）、2 M尿素、10 mM二硫苏糖醇（dithiothreitol, DTT）溶液中，以酶与蛋白质量比1:100的比例加入胰蛋白酶，于25℃酶解16小时。酶解反应通过酸化终止。采用疏水相互作用色谱（hydrophobic interaction chromatography, HILIC）按照厂商操作手册（美国马里兰州PolyLC公司）对血清肽段进行分级分离，额外设置溶剂梯度递减以洗脱亲水性逐渐增强的肽段。共制备5个分级组分（未结合组分、25%、50%、70%及100%顺序溶剂洗脱组分），后续……

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Microsoft Excel（原Microsoft XLS格式）
# 急性腰痛发作期间性别差异的血清蛋白质组学分析

本研究使用了“持续性腰痛转归研究（Understanding persistent Pain Where it ResiDes, UPWaRD）”纵向队列的亚样本，这些受试者在基线评估时提供了血清样本（临床试验注册号：ACTRN12619000002189）[1]。本研究共纳入59名有急性腰痛发作史的受试者，按年龄与性别进行匹配：女性30名（平均年龄42±15岁），男性29名（平均年龄38±16岁）。同时，我们从UPWaRD队列中纳入了数量匹配的、3个月时腰痛缓解或未缓解的受试者。具体而言，15名男性与15名女性受试者被归为腰痛缓解组（数字疼痛评分量表Numerical Rating Scale, NRS=0.30±0.47），而14名男性与15名女性受试者被归为未缓解组（NRS=4.0±1.7），该结果来自3个月随访评估。

使用Scaffold软件（版本4.8.7，美国俄勒冈州波特兰Proteome Software公司）通过光谱计数法对鸟枪蛋白质组学结果进行对比分析。肽段鉴定结果被接受……