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

Abstract

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.

Improving Selectivity and Sensitivity of Protein Quantitation By Lc–Hr–Ms/Ms: Determination of Somatropin in Rat Plasma

“…In recent years, the field of HRMS has seen considerable improvements with regard to sensitivity, linear dynamic range and scan modes, and the technique is increasingly seen as a serious option for quantitative bioanalysis, even though the instrument sensitivity of triple-quadrupole MS typically still is superior [14,15]. So far, most published applications of HRMS in quantitative bioanalysis have been for small molecules or peptides [16][17][18][19] and although the applicability of HRMS has also been demonstrated for protein quantification after digestion [20][21][22][23][24][25], this field clearly still is in its infancy and a systematic investigation of the potential of HRMS to improve selectivity is still lacking.…”

Effect of insulin treatment on circulating insulin‐like growth factor I and IGF‐binding proteins in cats with diabetes mellitus

“…A targeted mass spectrometry (MS)‐based method, previously validated for cats, was applied to determine the concentrations of IGF‐I, IGF‐II, IGFBP‐3, and IGFBP‐5 in 3 cats with DM later achieving remission. Analyses were performed in serum at T0 and T1 exactly as described by Sundberg et al Briefly, the serum proteins were digested with trypsin and isotopically labeled internal standards, 4 QPrESTs (Atlas antibodies, Stockholm, Sweden) and 1 synthetic peptide (New England Peptide, Gardner, Massachusetts), were added to the samples.…”

“…A targeted mass spectrometry (MS)‐based method, previously validated for cats, was applied to determine the concentrations of IGF‐I, IGF‐II, IGFBP‐3, and IGFBP‐5 in 3 cats with DM later achieving remission. Analyses were performed in serum at T0 and T1 exactly as described by Sundberg et al Briefly, the serum proteins were digested with trypsin and isotopically labeled internal standards, 4 QPrESTs (Atlas antibodies, Stockholm, Sweden) and 1 synthetic peptide (New England Peptide, Gardner, Massachusetts), were added to the samples. The tryptic peptides were separated in reversed phase on an EASY‐nLC 1000 system (EASY‐nLC 1000 system, ThermoFisher Scientific, Waltham, Massachusetts) and electrosprayed on‐line to a QExactive Plus Orbitrap mass spectrometer (QExactive Plus Orbitrap mass spectrometer, ThermoFisher Scientific, Waltham, Massachusetts) operating in parallel reaction monitoring mode.…”

Expression of dopamine D2 and D3 receptors in the human retina revealed by positron emission tomography and targeted mass spectrometry

“…Relative quantification of D 2 R and D 3 R peptides from schizophrenia patient and control samples was based on the sum of the area under the curves (AUC) of the top three most intense peptide fragments. The use of a subset of three to six fragment ions (with acceptable peak purity) for peptide quantitation has been previously suggested to yield more accurate results than the entire set (Gallien and Domon, 2015; Sundberg et al, 2016). Endogenous peptides were matched to heavy isotope peptides based on their similar retention times and transition patterns.…”