Sample Preparation for High-Throughput Urine Proteomics Using 96-Well Polyvinylidene Fluoride (PVDF) Membranes.
Biomarker
Bottom-up
Liquid chromatography mass spectrometry
PVDF
Proteomics
Urine
Journal
Advances in experimental medicine and biology
ISSN: 0065-2598
Titre abrégé: Adv Exp Med Biol
Pays: United States
ID NLM: 0121103
Informations de publication
Date de publication:
2021
2021
Historique:
entrez:
7
5
2021
pubmed:
8
5
2021
medline:
11
5
2021
Statut:
ppublish
Résumé
Proteomics analysis of urine samples allows for studying the impact of system perturbation. However, meaningful proteomics-based biomarker discovery projects often require the analysis of large patient cohorts with hundreds of samples to describe the biological variability. Thus, robust high-throughput sample processing methods are a prerequisite for clinical proteomics pipelines that minimize experimental bias due to individual sample processing methods. Herein we describe a high-throughput method for parallel 96-well plate-based processing of urine samples for subsequent LC/MS-based proteomic analyses. Protein digestion and subsequent sample processing steps are efficiently performed in 96-well polyvinylidene fluoride (PVDF) membrane plate allowing for the use of vacuum manifolds for rapid liquid transfer, and multichannel pipettes and/or liquid handing robots. In this chapter we make available a detailed step-by-step protocol for our 'MStern blotting' sample processing strategy applied to patient urine samples followed by mass spectrometry-based proteomics analysis. Subsequently, we provide an example application using minimal volume of urine samples (e.g. 150 μL) collected from children pre and post thoracotomy to identify the predominant sites of protein catabolism and aid in the design of therapies to ameliorate protein catabolism and breakdown during critical illness. Furthermore, we demonstrate how the systemic state is reflected in the urine as an easily obtainable, stable, and safe biofluid.
Identifiants
pubmed: 33959902
doi: 10.1007/978-3-030-63908-2_1
doi:
Substances chimiques
Polyvinyls
0
polyvinylidene fluoride
24937-79-9
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1-12Références
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