Well-Plate µFASP for Proteomic Analysis of Single Pancreatic Islets.
J Proteome Res
; 21(4): 1167-1174, 2022 04 01.
Article
em En
| MEDLINE
| ID: mdl-35293755
ABSTRACT
Filter-aided sample preparation (FASP) is widely used in bottom-up proteomics for tryptic digestion. However, the sample recovery yield of this method is limited by the amount of the starting material. While â¼100 ng of digested protein is sufficient for thorough protein identification, proteomic information gets lost with a protein content <10 µg due to incomplete peptide recovery from the filter. We developed and optimized a flexible well-plate µFASP device and protocol that is suitable for an â¼1 µg protein sample. In 1 µg of HeLa digest, we identified 1295 ± 10 proteins with µFASP followed by analysis with liquid chromatography-mass spectrometry. In contrast, only 524 ± 5 proteins were identified with the standard FASP protocol, while 1395 ± 4 proteins were identified in 20 µg after standard FASP as a benchmark. Furthermore, we conducted a combined peptidomic and proteomic study of single pancreatic islets with well-plate µFASP. Here, we separated neuropeptides and digested the remaining on-filter proteins for bottom-up proteomic analysis. Our results indicate inter-islet heterogeneity for the expression of proteins involved in glucose catabolism, pancreatic hormone processing, and secreted peptide hormones. We consider our method to provide a useful tool for proteomic characterization of samples where the biological material is scarce. All proteomic data are available under DOI 10.6019/PXD029039.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ilhotas Pancreáticas
/
Proteômica
Limite:
Humans
Idioma:
En
Revista:
J Proteome Res
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Suécia