Detection and Quantitation of Circulating Human Irisin by Tandem Mass Spectrometry

Abstract

Exercise provides many health benefits, including improved metabolism, cardiovascular health, and cognition. We have shown previously that FNDC5, a type I transmembrane protein, and its circulating form, irisin, convey some of these benefits in mice. However, recent reports questioned the existence of circulating human irisin both because human FNDC5 has a non-canonical ATA translation start and because of claims that many human irisin antibodies used in commercial ELISA kits lack required specificity. In this paper we have identified and quantitated human irisin in plasma using mass spectrometry with control peptides enriched with heavy stable isotopes as internal standards. This precise state-of-the-art method shows that human irisin is mainly translated from its non-canonical start codon and circulates at ∼ 3.6 ng/ml in sedentary individuals; this level is increased to ∼ 4.3 ng/ml in individuals undergoing aerobic interval training. These data unequivocally demonstrate that human irisin exists, circulates, and is regulated by exercise.

Figure 1. Analysis of Irisin Peptides by Mass Spectrometry

(A) Schematic representation of the FNDC5 protein structure (top) and irisin (bottom). SP = signal peptide, H = hydrophobic domain, C = c-terminal domain. Human FNDC5 sequence with corresponding domains colored. Human irisin sequence is underlined as well as synthetic AQUA peptides used in this study (red). (B) Immunoblotting of irisin plasma samples from three subjects undergoing aerobic interval training with or without deglycosylation enzyme (Protein Deglycosylation Mix (NEB)) and deglycosylated recombinant irisin. (C) MS2 spectra acquired using a Q Exactive mass spectrometer for the two synthetic AQUA peptides and their b-, y-ion series m/z values. Mass accuracy values are given in PPMs and “#” denotes the heavy valine residue. (D) PRM elution profile for the y-ions for the AQUA peptides using Skyline software. Retention times for each peptide are labeled on the x-axis and y-axis represents the relative intensity for each y-ion peak. See also Figure S1.

Figure 2. Detection of Irisin in Human Plasma

(A) SDS PAGE separation of 50 μg of plasma from each subject and visualized by coomassie staining. Molecular mass regions corresponding to completely deglycosylated irisin (10–15 kDa) were excised from six separate gels (300 μg from the original 100 μl plasma) for each subject and digested in-gel in the presence of 12.5 femtomoles of each internal standard AQUA peptide. (B) PRM elution profile for internal tryptic irisin peptide (FIQEVNTTR) using Skyline software found in sedentary subject 1. Top panel is the deamidated asparagine form of the peptide found in the plasma, middle panel is the unmodified peptide found in the plasma and the bottom panel is 12.5 femtomoles of heavy internal standard (IS) AQUA peptide. (C) Irisin levels in plasma from sedentary subjects (Sedentary) or subject undergoing aerobic interval training (Aerobic). Values are shown as mean±SEM. n=4 (Sedentary) and n=6 (Aerobic). * p= 0.0411 compared to sedentary subject group as determined by unpaired t-test, two-tailed. (D) Depicted are several plasma proteins and their circulating concentrations ranging from the μg/ml (red), ng/mL (yellow) and pg/ml (blue) levels. We quantify circulating plasma irisin at a 3–5 ng/ml. See also Figure S2.