Biomarkers of systemic inflammation in farmers with musculoskeletal disorders; a plasma proteomic study

The investigated subjects were selected from a prospective study cohort established with the intention to study health promoting factors related to farming and lifestyle [7]. All occupationally active male farmers born between 1930 and 1949 in nine rural municipalities in Sweden were identified in the Swedish National Farm Register and included in the cohort. The cohort represents a variety of farm types and geographical variation. The areas were chosen with consideration to known east-west and north-south cardiovascular disease gradients in the Swedish population [8] and to represent a variety of farm types and geographical variation over the country. Farmers were defined as men who owned or rented a farm and who spent at least 25 h per week farming. The occupational activity was checked with local representatives from the Federation of Swedish Farmers. To each farmer a rural referent, matched by age, sex and residential area, was sampled from the National Population Register. The rural referents were to be occupationally active in other than farming. Altogether 1220 farmers and 1130 rural non-farmers were eligible and included in the cohort. The farmers and the rural referents (n = 2350) were invited to an extensive health survey including questionnaires, interviews, physical examinations and laboratory test [9].

The cross-sectional population-based survey identified farmers with MSD using a questionnaire with a number of items addressing symptoms from back, hip, knee, neck, forearms and shoulders. Information on symptoms from the musculoskeletal system was assessed by self-administrated questionnaire [3]. The subjects were asked to indicate (yes or no) if they had ever (more than occasionally) had pain, aches or discomfort in the neck-shoulder area, the low back, hips or knees. The questionnaire file shows this in more detail (see Additional file 1). Similar symptoms during the last year were also indicated. The subjects were asked to indicate whether they had sought medical advice or treatment for the various symptoms, and concerning low back and knee symptoms, whether they had been on sick leave due to the illness. A large number of farmers and non-farmers (89 %) reported symptoms from musculoskeletal system. The farmers reported significantly more symptoms from low back and hips [4, 9].

This large material was used to identify farmers with symptoms from the musculoskeletal system only. Thirteen farmers working with swine confinement and cattle raising and their rural referents (Table 1) were selected and included in this explorative hypothesis generated study.

All samples were blinded before analysis. Plasma from farmers and referents were prepared at the same time and not separately. 50 μl of plasma was applied onto an albumin & IgG Depletion column (GE Healthcare). Albumin and IgG were removed according to the manufacturer’s recommendation. The samples were then desalted and lyophilized. The protein concentration was determined according to Bradford [10].

The lyophilized proteins were dissolved in 0.20 ml urea sample solution according to Görg [11]. 2-DE was performed in a horizontal 2-DE setup (IPGphore and Multiphore from GE Healthcare), as described in detail previously [12] and essentially according to Görg [11]. The samples (containing 50 μg protein for analytical gels and 900 μg for preparative gels) were applied by in-gel rehydration (according to the manufacturer’s instructions) for 12 h using low voltage (30 V) in pH 4–7 IPGs. The proteins were then focused for up to 32 000 Vhs at a maximum voltage of 8000 V. IPGs were either used immediately for second dimensional analysis, or stored at −70 °C until analyzed. The second dimension (SDS-PAGE) was carried out by transferring the proteins to gradient gels cast on GelBond PAG film (0.5/180/245 mm, 11–18%T, 1.5%C, 33–0 % glycerol) running at 30 mA and up to 1000 V for about 5 h. In analytical gels, separated proteins were detected by silver staining according to Schevchenko as described previously [12]. The preparative gels, for MS analysis, were fluorescently stained with SYPRO Ruby [12]. All staining and washing steps were performed with continuous gentle agitation. The protein patterns were analyzed as digitized images, using a CCD camera in combination with a computerized imaging 12-bit system designed for evaluation of 2-DE patterns. The amount of protein in a spot was assessed as background corrected optical density, integrated over all pixels in the spot and expressed as integrated optical density (IOD). In order to correct for differences in total silver stain intensity between different 2-DE images, the amounts of the compared protein spots were quantified as optical density for individual spot per total protein intensity of all spots in the same gel. Thereby ppm-values (parts per million) for all proteins were generated that were evaluated for differences between the groups.

Protein spots were excised using a homemade spot picker. The picked protein spots were digested with trypsin (Promega/SDS Biosciences, Falkenberg, Sweden) as described previously [13]. The trypsinated peptides were applied to mass spectrometry for protein identification. The dried tryptic samples from fluorescently stained proteins were dissolved in 6 μl of 0.1 % formic acid. Peptides were analyzed using an on-line nano-flow HPLC system (EASY-nLC; Proxeon, Bruker Daltonics) in conjugation with the mass spectrometer HCTultra PTM Discovery System (Bruker Daltonics) as previously described [13]. LC-MS/MS spectra were processed by Bruker Daltonics DataAnalysis 3.4 (Bruker Daltonics, Bremen, Germany) and resulting MS/MS data were searched in NCBInr and Swiss-Prot database on MASCOT server: www.​matrixscience.​com Database search parameters were set as follows: the enzyme trypsin was used; up to one missed cleavage was allowed; fixed modification included were carbamidomethylation of cysteine and oxidation of methionine; mass tolerance for MS precursor ion was 0.8 Da and for MS/MS fragment ion was 0.6 Da; and charge states were varied. Criteria for identification of a protein were at least 3 peptides of the protein should be identified with a MASCOT score over 25 and an expectation value <1. The mass list generated from the major peaks of the MALDI spectra was submitted to a database search (NCBI or SWISS-PROT) using MS-FIT search engines. Restrictions were placed on species (Human), mass tolerance (50 ppm), maximum missed cleavages by trypsin (up to 1) and cysteine modification by carbamidomethylation.

Non-parametric Mann-Whitney U test was used as statistical method to calculate significant differences between groups. A p < 0.05 was considered statistically significant. All results are given as mean ± standard deviation. IBM SPSS Statistics 20 was used as statistical software.

To determine proper 2DE conditions for the analysis to be made, plasma proteins were separated in the pI ranges 3–10 and 4–7 (first dimension) followed by SDS-PAGE (second dimension). As illustrated in Fig. 1, most of the spots detected in pI range 3–10 were also detected in the pI range 4–7 (about 90 %). The marked areas in Fig. 1 illustrate the good resolving power when using IPGs in pI range 4–7 compared to 3–10. A few proteins (Fig. 1a, dashed squares9 with pI outside the 4–7 range were lost. Due to the good separation in the pI range 4–7 we decided to analyze all samples using IPGs 4–7. Mapping of the plasma proteome in the pI range 4–7 was performed by mass spectrometry. Seventy five spots could be identified (Table 2, Fig. 2). According to the accession number these identified protein spots represent 20 different proteins. Several of the identified proteins were expressed as different isoforms due to post- translational modifications such as glycosylation and phosphorylation.

More than 200 protein spots were detected in all samples. To investigate possible differences in the plasma proteomes of farmers with MSD and rural referents, protein spots present in at least 50 % of the gels in either group were matched, quantified for intensity, and compared between the groups. Proteins whose concentrations were statistically different in the two groups were then identified using mass spectrometry. As shown in Table 3, the levels of leucine-rich alpha-2-glycoprotein, haptoglobin, complement factor B, serotransferrin, one isoform of kininogen (spot1507), one isoform of alpha-1-antitrypsin (spot 2404), and two isoforms of hemopexin (spots 6603 and 7602) were higher in farmers with MSD than in rural referents. On the other hand, the levels of alpha-2-HS-glycoprotein, alpha-1B-glycoprotein, vitamin D- binding protein, apolipoprotein A1, antithrombin, one isoform of kininogen (spot 2607), and one isoform of alpha-1-antitrypsin (spot 3802) were lower (Table 2). Figure 3 pinpoints the statistically differing proteins on a 2DE gel, while Fig. 4 illustrates how the different isoforms of kininogen, alpha-1-antitrypsin, and hemopexin were quantitatively expressed in farmers with MSD and referents, respectively.