Research Article: Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry

Date Published: December 1, 2016

Publisher: Public Library of Science

Author(s): Mårten Sundberg, Emma M. Strage, Jonas Bergquist, Bodil S. Holst, Margareta Ramström, René P. Zahedi.


Today immunoassays are widely used in veterinary medicine, but lack of species specific assays often necessitates the use of assays developed for human applications. Mass spectrometry (MS) is an attractive alternative due to high specificity and versatility, allowing for species-independent analysis. Targeted MS-based quantification methods are valuable complements to large scale shotgun analysis. A method referred to as parallel reaction monitoring (PRM), implemented on Orbitrap MS, has lately been presented as an excellent alternative to more traditional selected reaction monitoring/multiple reaction monitoring (SRM/MRM) methods. The insulin-like growth factor (IGF)-system is not well described in the cat but there are indications of important differences between cats and humans. In feline medicine IGF–I is mainly analyzed for diagnosis of growth hormone disorders but also for research, while the other proteins in the IGF-system are not routinely analyzed within clinical practice. Here, a PRM method for quantification of IGF–I, IGF–II, IGF binding protein (BP) –3 and IGFBP–5 in feline serum is presented. Selective quantification was supported by the use of a newly launched internal standard named QPrEST™. Homology searches demonstrated the possibility to use this standard of human origin for quantification of the targeted feline proteins. Excellent quantitative sensitivity at the attomol/μL (pM) level and selectivity were obtained. As the presented approach is very generic we show that high resolution mass spectrometry in combination with PRM and QPrEST™ internal standards is a versatile tool for protein quantitation across multispecies.

Partial Text

Insulin-like growth factor–I and–II (IGF–I and–II) are important regulators of growth and metabolism in both humans and animals. In adult humans almost all IGFs are bound to insulin-like growth factor binding proteins (IGFBP 1–6) which in addition to modulating IGF-bioavailability exert their own biological effects [1,2]. In adult humans, IGFs circulate mainly as ternary complexes bound to IGFBP–3 or IGFBP–5 and the acid labile subunit (IGFALS). The ternary complex prolongs the half-life of IGFs and thus is a major determinant of IGF-concentrations [2]. The IGF-system is not well described in the cat, but there are indications of important differences between cats and humans [3]. To understand the IGF–system in the cat, and to improve clinical diagnoses, it would be beneficial to be able to measure IGF–I,–II and IGFBPs. In feline medicine IGF–I is mainly analyzed for diagnosis of growth hormone disorders but also for research [4]. IGF–II, IGFBP–3 and –5 are not routinely analyzed in clinical practice and there are to our knowledge no commercial validated assays for cats. Large variability for human IGF-I immunoassays has been reported [5]. The College of American Pathologists (CAP) proficiency testing program for IGF–I demonstrated an interlaboratory variability of up to 34% CV from September 2011 to March 2013, while a mass spectrometry-based method demonstrated better reproducibility (CV<16%) [6]. One study demonstrated large variability when measuring feline IGF-I with four non cat-specific immunoassays [7]. Applying human immunoassays for analysis of samples from other species might cause problems including no or weak reactivity or unwanted cross-reactivity, and the assays therefore need to be thoroughly validated before use. Measurements of IGFs with immunoassays are difficult due to interference of IGFBPs, which may cause both false high and false low values depending on assay [8]. In this study, a PRM-method was set up for quantification of four feline growth related hormones (IGF–I,–II, IGFBP–3 and –5). There are no previously established methods to measure feline IGF-II, IGFBP–3 or IGFBP–5. To our knowledge, the present method is the first one developed for quantifying all four proteins in cat serum. One advantage of the MS-based method over immunoassays is that all target proteins are measured within the same experimental run. This reduces both variation due to methodology and the amount of serum needed for analysis, which can be a great advantage in cats and other small animals.   Source: