Optimization of Synovial Fluid Collection and Processing for NMR Metabolomics and LC-MS/MS Proteomics

Abstract

Synovial fluid (SF) is of great interest for the investigation of orthopedic pathologies, as it is in close proximity to various tissues that are primarily altered during these disease processes and can be collected using minimally invasive protocols. Multi-"omic" approaches are commonplace, although little consideration is often given for multiple analysis techniques at sample collection. Nuclear magnetic resonance (NMR) metabolomics and liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics are two complementary techniques particularly suited to the study of SF. However, currently there are no agreed upon standard protocols that are published for SF collection and processing for use with NMR metabolomic analysis. Furthermore, the large protein concentration dynamic range present within SF can mask the detection of lower abundance proteins in proteomics. While combinational ligand libraries (ProteoMiner columns) have been developed to reduce this dynamic range, their reproducibility when used in conjunction with SF, or on-bead protein digestion protocols, has yet to be investigated. Here we employ optimized protocols for the collection, processing, and storage of SF for NMR metabolite analysis and LC-MS/MS proteome analysis, including a Lys-C endopeptidase digestion step prior to tryptic digestion, which increased the number of protein identifications and improved reproducibility for on-bead ProteoMiner digestion.

Keywords: Lys-C endopeptidase; mass spectrometry; metabolomics; nuclear magnetic resonance; proteomics; synovial fluid.

Figure 1

Optimization of equine synovial fluid processing for 1D ¹H NMR metabolome analysis. The reproducibility of the metabolome for different processing protocols for equine synovial fluid (SF) was assessed using principal component analysis (PCA). These protocols included (A) with (n = 6) and without (n = 8) a centrifugation step (2540g and 4 °C for 5 min) prior to freezing and (B) the use of different freezing methods (−20 °C (n = 7), −80 °C (n = 6), dry ice (n = 5), and liquid nitrogen (n = 7)). (C) PCA showing reproducibility of the finalized SF processing method (including centrifugation and liquid nitrogen snap freezing) using three separate equine donors with three technical replicates for each donor. PCA shaded regions depict 95% confidence regions.

Figure 2

ProteoMiner column loading of synovial fluid. (A) Protein profiles of on-bead digests and flow-through (FT) following 1, 2.5, and 5 mg protein loading. (B) Number of proteins identified via LC-MS/MS following bead enrichment of depleted proteins and column flow-through for 1 mg, 2.5 mg and 5 mg protein loadings, and (C) 5 mg and 10 mg protein loadings for another set of pooled synovial fluid. A full protein gel image can be found in Figure S5.

Figure 3

Protein profiles of native and ProteoMiner processed equine synovial fluid following protein digestion. (A) Different ProteoMiner loading and digestion protocols ± Lys-C endopeptidase predigestion (yellow boxes indicate profiles including Lys-C predigestion, arrow indicates a protein band not present following Lys-C predigestion protocols). (B) Trypsin digestion protocols for native and ProteoMiner processed synovial fluid. (C) ProteoMiner bead protein profiles following digestion protocols (red box indicates last stages of digestion were carried out in-solution, not on the beads). Full protein gel images can be found in Figure S8.

Figure 4

Number of Mascot protein identifications for native and ProteoMiner processed equine synovial fluid following different loading and protein digestion protocols involving trypsin ± Lys-C endopeptidase predigestion. Pilot study, n = 1/digestion protocol.

Figure 5

Reproducibility of ProteoMiner processed synovial fluid protein digests with and without Lys-C endopeptidase predigestion. Tryptic peptide reproducibility of three technical replicates (A) with and (B) without a 4 h Lys-C predigestion prior to 16 h + 2 h on-bead trypsin digestion. (C) Average number of missed cleavages per peptide, and (D) principal component analysis (PCA) of tryptic peptide profiles with (green) and without (red) Lys-C predigestion for a series of trypsin digestion protocols. Peptide abundances were analyzed via LC-MS/MS with a 1 h LC gradient. CV = coefficient of variation. **p < 0.01.