Development of an SRM method for absolute quantitation of MYDGF/C19orf10 protein

• Published in: Proteomics. Clinical applications Vol. 10; no. 6; pp. 663 - 670
• Main Authors: Dwivedi, Ravi C., Krokhin, Oleg V., El-Gabalawy, Hani S., Wilkins, John A.
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01.06.2016; Wiley Subscription Services, Inc
• Subjects: Albumins - chemistry; Amino Acid Sequence; Arthritis, Rheumatoid - blood; Arthritis, Rheumatoid - diagnosis; Arthritis, Rheumatoid - pathology; Biological Assay - methods; Biological Assay - standards; C19orf10; Carbon Isotopes; Chemical Precipitation; Humans; Immunoglobulin G - chemistry; Interleukins - blood; Interleukins - isolation & purification; Isotope Labeling - methods; Limit of Detection; Mass Spectrometry - methods; Mass Spectrometry - standards; MYDGF; Nitrogen Isotopes; Peptides; Peptides - analysis; Peptides - chemistry; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Serum; SRM; Synovial fluid; Synovial Fluid - chemistry; MYDGF; Synovial fluid; Serum; C19orf10; SRM
• ISSN: 1862-8346; 1862-8354
• DOI: 10.1002/prca.201500057

Purpose To develop a MS‐based selected reaction monitoring (SRM) assay for quantitation of myeloid‐derived growth factor (MYDGF) formerly chromosome 19 open reading frame (C19orf10). Experimental design Candidate reporter peptides were identified in digests of recombinant MYDGF. Isotopically labeled forms of these reporter peptides were employed as internal standards for assay development. Two reference peptides were selected SYLYFQTFFK and GAEIEYAMAYSK with respective LOQ of 42 and 380 attomole per injection. Results Application of the assay to human serum and synovial fluid determined that the assay sensitivity was reduced and quantitation was not achievable. However, the partial depletion of albumin and immunoglobulin from synovial fluids provided estimates of 300–650 femtomoles per injection (0.7–1.6 nanomolar (nM) fluid concentrations) in three of the six samples analyzed. Conclusions and clinical relevance A validated sensitive assay for the quantitation of MYDGF in biological fluids was developed. However, the endogenous levels of MYDGF in such fluids are at or below the current levels of quantitation. The levels of MYDGF are lower than those previously reported using an ELISA. The current results suggest that additional steps may be required to remove high abundance proteins or to enrich MYDGF for SRM‐based quantitation.