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A Novel Cell Fixation Method that Greatly Enhances Protein Identification in Microproteomic Studies Using Laser Capture Microdissection and Mass Spectrometry
Author(s) -
Gordon Ana,
Kannan Shravan Kumar,
Gousset Karine
Publication year - 2018
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201700294
Subject(s) - laser capture microdissection , mass spectrometry , proteomics , chemistry , microdissection , chromatography , mass spectrometry imaging , biochemistry , gene expression , gene
Microproteomic studies have improved our knowledge of cell biology. Yet, with mass spectrometry (MS) analysis, accuracy can be lost for protein identification and quantification when using heterogeneous samples. Laser capture microdissection (LCM) allows for the enrichment of specific subsets of cells to study their proteome; however, sample fixation is necessary. Unfortunately, fixation hampers MS results due to protein cross‐linking. The aim of this study was to identify both a fixation protocol and an extraction method that returns the best yield of proteins for downstream MS analysis, while preserving cellular structures. We compared glutaraldehyde (GLU), a common fixative to preserve cells, to dithiobispropionimidate (DTBP), a cleavable cross‐linker. Our DTBP fixation/extraction protocol greatly increased the protein recovery. In fact, while 1000 GLU fixed cells returned only 159 unique protein hits, from 1464 unique peptides of 1994 unique collected spectra, 1000 DTBP fixed cells resulted in 567 unique collected protein hits, from 7542 unique peptides, of 10,401 unique collected spectra. That is, a 3.57‐fold increase in protein hits, 5.15‐fold increase in unique peptides, and a 5.22‐fold increase in unique collected spectra. Overall, the novel protocol introduced here allows for a very efficient protein recovery and good sample quality for MS after sample collection using LCM.