Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry

Background: Peptide Tyr-Tyr (PYY1-36), pancreatic polypeptide (PP1-36) and neuropeptide Y (NPY1-36) constitute the PP-fold family of peptides that is involved in metabolic regulation. Very low plasma concentrations and cleavage into active 3-36 fragments challenge bioanalytical assays used for the q...

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Main Authors: Bally, L. (Author), Eugster, P.J (Author), Grouzmann, E. (Author), Herzig, D. (Author), Kuenzli, C. (Author), Nakas, C.T (Author), Reverter-Branchat, G. (Author), Rindlisbacher, B. (Author), Stauffer, T. (Author)
Format: Article
Language:English
Published: Oxford University Press 2022
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Online Access:View Fulltext in Publisher
LEADER 02897nam a2200313Ia 4500
001 10-1093-clinchem-hvab229
008 220425s2022 CNT 000 0 und d
020 |a 00099147 (ISSN) 
245 1 0 |a Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry 
260 0 |b Oxford University Press  |c 2022 
300 |a 11 
856 |z View Fulltext in Publisher  |u https://doi.org/10.1093/clinchem/hvab229 
520 3 |a Background: Peptide Tyr-Tyr (PYY1-36), pancreatic polypeptide (PP1-36) and neuropeptide Y (NPY1-36) constitute the PP-fold family of peptides that is involved in metabolic regulation. Very low plasma concentrations and cleavage into active 3-36 fragments challenge bioanalytical assays used for the quantification of these peptides. Methods: We developed a multiplexed isotopic dilution assay to quantify PYY1-36, PP1-36, and NPY1-36 and their dipeptidyl peptidase-4 (DPP4)-derived metabolites PYY3-36, PP3-36 and NPY3-36. All peptides were immunocaptured from plasma using a monoclonal antibody and quantified by micro-ultra-HPLC-MS/MS. Blood samples from healthy volunteers were collected fasting and 30 min after nutrient stimulation. Method comparison was performed with commercial immunoassays. Results: Linearity was shown in the measured intervals (r2 > 0.99). The lower limit of quantification (LLOQ) with a CV at 20% was 1.5 pM for PYY1-36 and PYY3-36, 3.0 pM for PP1-36 and PP3-36, 0.8 pM for NPY1-36 and 0.5 pM for NPY3-36. In all cases, intra- and inter-assay bias and imprecision were <21%. Pre-analytical stability required addition of a protease inhibitor cocktail. Physiological concentrations of PYY3-36, NPY3-36, PP1-36 and PP3-36 were above the LLOQ in 43% to 100% of the samples. PYY1-36 and NPY1-36 were above the LLOQ in 9% and 0% of the samples, respectively. Immunoassays showed higher concentrations of measurands and poor agreement when compared with micro-UHPLC-MS/MS. Conclusions: The assay allowed for specific multiplexed analysis of the PP-fold family of peptides and their DPP4-cleaved fragments in a single sample, thereby offering new perspectives to study the role and therapeutic potential of these essential peptide hormones in health and metabolic disease. © 2022 American Association for Clinical Chemistry 2022. 
650 0 4 |a DPP4 
650 0 4 |a immunoassay 
650 0 4 |a micro-UHPLC-MS/MS 
650 0 4 |a neuropeptide Y (NPY) 
650 0 4 |a pancreatic polypeptide (PP) 
650 0 4 |a peptide YY (PYY) 
700 1 |a Bally, L.  |e author 
700 1 |a Eugster, P.J.  |e author 
700 1 |a Grouzmann, E.  |e author 
700 1 |a Herzig, D.  |e author 
700 1 |a Kuenzli, C.  |e author 
700 1 |a Nakas, C.T.  |e author 
700 1 |a Reverter-Branchat, G.  |e author 
700 1 |a Rindlisbacher, B.  |e author 
700 1 |a Stauffer, T.  |e author 
773 |t Clinical Chemistry