Cargando…
Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model
Suspension TRAPping filter (sTRAP) is an attractive sample preparation method for proteomics studies. The sTRAP protocol uses 5% SDS that maximizes protein solubilization. Proteins are trapped on a borosilicate glass membrane filter, where SDS is subsequently removed from the filter. After trypsin d...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10177283/ https://www.ncbi.nlm.nih.gov/pubmed/37174641 http://dx.doi.org/10.3390/cells12091242 |
_version_ | 1785040602115604480 |
---|---|
author | Thanou, Evangelia Koopmans, Frank Pita-Illobre, Débora Klaassen, Remco V. Özer, Berna Charalampopoulos, Ioannis Smit, August B. Li, Ka Wan |
author_facet | Thanou, Evangelia Koopmans, Frank Pita-Illobre, Débora Klaassen, Remco V. Özer, Berna Charalampopoulos, Ioannis Smit, August B. Li, Ka Wan |
author_sort | Thanou, Evangelia |
collection | PubMed |
description | Suspension TRAPping filter (sTRAP) is an attractive sample preparation method for proteomics studies. The sTRAP protocol uses 5% SDS that maximizes protein solubilization. Proteins are trapped on a borosilicate glass membrane filter, where SDS is subsequently removed from the filter. After trypsin digestion, peptides are analyzed directly by LC-MS. Here, we demonstrated the use of a low-cost plasmid DNA micro-spin column for the sTRAP sample preparation of a dilution series of a synapse-enriched sample with a range of 10–0.3 µg. With 120 ng tryptic peptides loaded onto the Evosep LC system coupled to timsTOF Pro 2 mass spectrometer, we identified 5700 protein groups with 4% coefficient of variation (CoV). Comparing other sample preparation protocols, such as the in-gel digestion and the commercial Protifi S-TRAP with the plasmid DNA micro-spin column, the last is superior in both protein and peptide identification numbers and CoV. We applied sTRAP for the analysis of the hippocampal proteome from the 5xFAD mouse model of Alzheimer’s disease and their wildtype littermates, and revealed 121 up- and 54 down-regulated proteins. Protein changes in the mutant mice point to the alteration of processes related to the immune system and Amyloid aggregation, which correlates well with the known major Alzheimer’s-disease-related pathology. Data are available via ProteomeXchange with the identifier PXD041045. |
format | Online Article Text |
id | pubmed-10177283 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-101772832023-05-13 Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model Thanou, Evangelia Koopmans, Frank Pita-Illobre, Débora Klaassen, Remco V. Özer, Berna Charalampopoulos, Ioannis Smit, August B. Li, Ka Wan Cells Article Suspension TRAPping filter (sTRAP) is an attractive sample preparation method for proteomics studies. The sTRAP protocol uses 5% SDS that maximizes protein solubilization. Proteins are trapped on a borosilicate glass membrane filter, where SDS is subsequently removed from the filter. After trypsin digestion, peptides are analyzed directly by LC-MS. Here, we demonstrated the use of a low-cost plasmid DNA micro-spin column for the sTRAP sample preparation of a dilution series of a synapse-enriched sample with a range of 10–0.3 µg. With 120 ng tryptic peptides loaded onto the Evosep LC system coupled to timsTOF Pro 2 mass spectrometer, we identified 5700 protein groups with 4% coefficient of variation (CoV). Comparing other sample preparation protocols, such as the in-gel digestion and the commercial Protifi S-TRAP with the plasmid DNA micro-spin column, the last is superior in both protein and peptide identification numbers and CoV. We applied sTRAP for the analysis of the hippocampal proteome from the 5xFAD mouse model of Alzheimer’s disease and their wildtype littermates, and revealed 121 up- and 54 down-regulated proteins. Protein changes in the mutant mice point to the alteration of processes related to the immune system and Amyloid aggregation, which correlates well with the known major Alzheimer’s-disease-related pathology. Data are available via ProteomeXchange with the identifier PXD041045. MDPI 2023-04-25 /pmc/articles/PMC10177283/ /pubmed/37174641 http://dx.doi.org/10.3390/cells12091242 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Thanou, Evangelia Koopmans, Frank Pita-Illobre, Débora Klaassen, Remco V. Özer, Berna Charalampopoulos, Ioannis Smit, August B. Li, Ka Wan Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model |
title | Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model |
title_full | Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model |
title_fullStr | Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model |
title_full_unstemmed | Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model |
title_short | Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer’s Disease Mouse Model |
title_sort | suspension trapping filter (strap) sample preparation for quantitative proteomics in the low µg input range using a plasmid dna micro-spin column: analysis of the hippocampus from the 5xfad alzheimer’s disease mouse model |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10177283/ https://www.ncbi.nlm.nih.gov/pubmed/37174641 http://dx.doi.org/10.3390/cells12091242 |
work_keys_str_mv | AT thanouevangelia suspensiontrappingfilterstrapsamplepreparationforquantitativeproteomicsinthelowμginputrangeusingaplasmiddnamicrospincolumnanalysisofthehippocampusfromthe5xfadalzheimersdiseasemousemodel AT koopmansfrank suspensiontrappingfilterstrapsamplepreparationforquantitativeproteomicsinthelowμginputrangeusingaplasmiddnamicrospincolumnanalysisofthehippocampusfromthe5xfadalzheimersdiseasemousemodel AT pitaillobredebora suspensiontrappingfilterstrapsamplepreparationforquantitativeproteomicsinthelowμginputrangeusingaplasmiddnamicrospincolumnanalysisofthehippocampusfromthe5xfadalzheimersdiseasemousemodel AT klaassenremcov suspensiontrappingfilterstrapsamplepreparationforquantitativeproteomicsinthelowμginputrangeusingaplasmiddnamicrospincolumnanalysisofthehippocampusfromthe5xfadalzheimersdiseasemousemodel AT ozerberna suspensiontrappingfilterstrapsamplepreparationforquantitativeproteomicsinthelowμginputrangeusingaplasmiddnamicrospincolumnanalysisofthehippocampusfromthe5xfadalzheimersdiseasemousemodel AT charalampopoulosioannis suspensiontrappingfilterstrapsamplepreparationforquantitativeproteomicsinthelowμginputrangeusingaplasmiddnamicrospincolumnanalysisofthehippocampusfromthe5xfadalzheimersdiseasemousemodel AT smitaugustb suspensiontrappingfilterstrapsamplepreparationforquantitativeproteomicsinthelowμginputrangeusingaplasmiddnamicrospincolumnanalysisofthehippocampusfromthe5xfadalzheimersdiseasemousemodel AT likawan suspensiontrappingfilterstrapsamplepreparationforquantitativeproteomicsinthelowμginputrangeusingaplasmiddnamicrospincolumnanalysisofthehippocampusfromthe5xfadalzheimersdiseasemousemodel |