iTraQ and SRM Verifies Potential Biomarkers in Multiple Sclerosis

In its early stages, multiple sclerosis (MS) is difficult to diagnose. Proteomic experiments have identified possible candidate biomarkers; however, few of these biomarkers can be verified.¹ In a recent publication, Kroksveen et al.¹ explore quantitative proteomic techniques using selected reaction monitoring (SRM) and iTRAQ to detect CSF proteins with differential abundance in patients with MS.²

MS is known to be initiated by a single demyelinating event, called a clinically isolated syndrome (CIS).³ For their experiments, Kroksveen et al.¹ obtained CSF samples from patients categorized as having relapsing remitting MS (RRMS), with and without CIS at the time of lumbar puncture. Samples were also obtained from patients with CIS that did not develop multiple sclerosis in the follow-up period. Control groups included patients with other inflammatory neurological diseases, patients with other neurological diseases, and spinal anesthesia subjects without any neurological symptoms.

The entire amount of protein in each depleted sample was reduced, alkylated, digested with trypsin, and iTRAQ labeled. Next, strong cation exchange (SCX) chromatography was performed on an Agilent 1100 series liquid chromatography system (Agilent). This system was coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific).

BLAST searches identified unique peptides for SRM analysis. CSF samples were reduced, alkylated, and digested with trypsin. Stable isotope-labeled internal standards (SISs) corresponding to the signature peptides were purchased in crude quality from Thermo Scientific and JPT Technologies. SRM analysis was performed on a Q-Trap 5500 (AB SCIEX) coupled to a Thermo Scientific Dionex Ultimate NCS-3500RS liquid chromatography system.

Results of these experiments identified a total of 1291 proteins, and more than 600 proteins were quantified in each comparison. Five proteins contained a fold change ≥ 2 and p value ≤ 0.05 and were significantly differentially abundant compared with MS patients and controls. These proteins were identified as alpha-1-antichymotrypsin, contactin-1, apolipoprotein D, clusterin, and kallikrein-6. While the Kroksveen group maintains that none of the verified proteins individually could differentiate between MS and controls, these proteins do warrant further investigation, as they are likely part of a larger panel of biomarkers.

References

1. Kroksveen, A.C., et al. (2011) ‘Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics‘, Journal of Proteomics, 74 (4), (pp. 371-388)

2. Kroksveen, A.C., et al. (2013) ‘Discovery and initial verification of differentially abundant proteins between multiple sclerosis patients and controls using iTRAQ and SID-SRM‘, Journal of Proteomics, 78, (pp. 312-325)

3. Miller, D., et al. (2005) ‘Clinically isolated syndromes suggestive of multiple sclerosis, part I: natural history, pathogenesis, diagnosis, and prognosis‘, Lancet Neurology, 4 (5), (pp. 281-288)