Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry
© American Association for Clinical Chemistry 2022..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:68 |
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| Enthalten in: |
Clinical chemistry - 68(2022), 4 vom: 31. März, Seite 584-594 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Reverter-Branchat, Gemma [VerfasserIn] |
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| Links: |
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| Themen: |
59763-91-6 |
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| Anmerkungen: |
Date Completed 06.04.2022 Date Revised 12.07.2022 published: Print Citation Status MEDLINE |
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| doi: |
10.1093/clinchem/hvab229 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM33550874X |
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| 100 | 1 | |a Reverter-Branchat, Gemma |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry |
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| 500 | |a Date Revised 12.07.2022 | ||
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| 520 | |a © American Association for Clinical Chemistry 2022. | ||
| 520 | |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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a DPP4 | |
| 650 | 4 | |a immunoassay | |
| 650 | 4 | |a micro-UHPLC-MS/MS | |
| 650 | 4 | |a neuropeptide Y (NPY) | |
| 650 | 4 | |a pancreatic polypeptide (PP) | |
| 650 | 4 | |a peptide YY (PYY) | |
| 650 | 7 | |a Neuropeptide Y |2 NLM | |
| 650 | 7 | |a Pancreatic Polypeptide |2 NLM | |
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| 700 | 1 | |a Eugster, Philippe J |e verfasserin |4 aut | |
| 700 | 1 | |a Kuenzli, Christina |e verfasserin |4 aut | |
| 700 | 1 | |a Rindlisbacher, Barbara |e verfasserin |4 aut | |
| 700 | 1 | |a Stauffer, Thomas |e verfasserin |4 aut | |
| 700 | 1 | |a Nakas, Christos T |e verfasserin |4 aut | |
| 700 | 1 | |a Herzig, David |e verfasserin |4 aut | |
| 700 | 1 | |a Grouzmann, Eric |e verfasserin |4 aut | |
| 700 | 1 | |a Bally, Lia |e verfasserin |4 aut | |
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