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

Reverter-Branchat, Gemma; Eugster, Philippe J; Kuenzli, Christina; Rindlisbacher, Barbara; Stauffer, Thomas; Nakas, Christos T; Herzig, David; Grouzmann, Eric; Bally, Lia (2022). Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry. Clinical chemistry, 68(4), pp. 584-594. Oxford University Press 10.1093/clinchem/hvab229

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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.


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.


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.


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.

Item Type:

Journal Article (Original Article)


04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Institute of Clinical Chemistry
04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Endocrinology, Diabetology and Clinical Nutrition

UniBE Contributor:

Reverter Branchat, Gemma, Nakas, Christos T., Herzig, David, Bally, Lia Claudia


600 Technology > 610 Medicine & health




Oxford University Press




Laura Goetschi

Date Deposited:

22 Feb 2022 17:22

Last Modified:

16 Dec 2022 18:38

Publisher DOI:


PubMed ID:


Uncontrolled Keywords:

DPP4 immunoassay micro-UHPLC-MS/MS neuropeptide Y (NPY) pancreatic polypeptide (PP) peptide YY (PYY)




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