Native chromatographic sample preparation of serum, plasma and cerebrospinal fluid does not comprise a risk for proteolytic biomarker loss.

We recently developed a native multidimensional chromatographic method for serum and plasma fractionation for proteomic biomarker search. This method has several advantages:parallelization and automation, high reproducibility and proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, optional enzymatic and immunological analytics additional to mass spectrometry, retaining metabolites, and information on complex formation, modification, and fragmentation of constituents. Nevertheless, native conditions have the probable risk of proteome alteration and biomarker loss by intrinsic proteinases. Hence, we tried to quantify here intrinsic proteolytic activity in native samples and fractions from serum, plasma and cerebrospinal fluid, as well as the effectiveness of intrinsic anti-proteinases during sample handling and preparation under our fractionation conditions. Therefore, we used several quantitative measures: (1) total proportion of intrinsic protein and peptide fractions, (2) azocasein hydrolysis and (3) mass spectrometric protein coverage and peptide numbers. To 1: In all non-fractionated specimens, neither decrease of protein concentration or molecular weight nor increase of peptide concentration was found after variable clotting or pre-incubation time. To 2: No azocasein hydrolysis was seen in these samples when prepared within a few hours at room temperature. Trypsin, when added in concentrations not higher than 0.85 µg/mL (0.04 µM), even was completely inhibited. Moreover, in native 1-D fractions no proteinase activity could be observed. To 3: Mass spectrometry confirmed that neither protein coverage nor peptide numbers differ significantly in 1-D or 2-D fractions after variable incubation time. These results suggest that intrinsic, native proteinase inhibitors potentially protect the proteomes considered, enabling "top-down" proteomic approaches under native conditions with serum, plasma and cerebrospinal fluid.

Higher levels of antibodies against the psoriasis-associated antigen pso p27 in cerebrospinal fluid from patients with low back pain and sciatica.

STUDY DESIGN: A prospective study comparing the presence of antibodies against the psoriasis-associated antigen pso p27 in pain-free control subjects and patients with low back pain and/or sciatica. OBJECTIVES: To analyze the amount of local inflammation present in human lumbar disc disorders, using anti-pso p27 antibodies in the cerebrospinal fluid as a marker and to analyze whether pain intensity correlates with this marker of inflammation. SUMMARY OF BACKGROUND DATA: Pso p27 is a major antigen in psoriasis that is also present, mostly locally, in other inflammatory disorders, such as sarcoidosis, inflammatory bowel disease, and ankylosing spondylitis, inflammation is also thought to play a major role in the generation of lumbar and radicular pain in degenerative disc disorders. METHODS: Anti-pso p27 antibodies in cerebrospinal fluid were quantified using an indirect enzyme-linked immunosorbent assay with pso p27 obtained from patients with psoriasis for use as an antigen. Fifteen patients with spinal stenosis, 11 patients without myelographic disc herniation, 17 patients with disc herniation, and 24 pain-free patient control subjects were studied. RESULTS: Significantly higher levels of anti-pso p27 antibodies were found in patients with myelographic signs of disc herniation than in with patients with no signs of herniation, patients with spinal stenosis, and control subjects. Patients with no known signs of disc herniation and patients with myelographic signs of spinal stenosis (< 10 mm in diameter) caused by degenerative changes, had higher levels of anti-pso p27 antibodies than did control subjects. However, these differences reached only borderline statistical significance. CONCLUSIONS: The results support those in previous reports, that inflammation probably plays an important role in degenerative disk disorders, particularly in disk herniations. That there was no correlation between pain intensity and anti-pso p27 activity indicates that the antigen is probably not essential in pain generation per se. The results may indicate that pso p27 is expressed secondary to, not as an initiator of, inflammation.