Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix-assisted laser desorption/ionisation-time of flight/time of flight-mass spectrometry.
Author(s): Umar A, Dalebout JC, Timmermans AM, Foekens JA, Luider TM
Publication: Proteomics, 2005, Vol. 5, Page 2680
PubMed ID: 15892168 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to evaluate the utility of four different denaturing solutions in preparation of laser-microdissected cells for MADLI-TOF-MS assessment. Specimens were frozen and post-fixed with ethanol.
Summary of Findings:
The authors report that few peptide peaks were detected when whole section or laser microdissected samples were prepared in urea or trifluoroethanol/NH4HCO3 buffers. Those prepared in RapiGest or SDS produced better results; and the ratio of signal to noise was best using SDS. Importantly, little overlap was observed among peaks when different denaturing buffers were used, suggesting buffer-specific results may be complimentary to one another. Prior preparation of whole-section samples on a monolithic HPLC column increased the number of peaks detected four-fold, while fractionation did not increase the number of peaks in microdissected samples.
Biospecimens
Preservative Types
- Frozen
- Ethanol
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Method of fluid acquisition Whole section
Laser microdissected cells
Biospecimen Acquisition Type of collection container/solution Microfuge tube
LoBind Eppendorf tubes
Analyte Extraction and Purification Analyte isolation method Fractionation on monolithic HPLC column
No fractionation
MALDI-TOF MS Specific Reaction solution Urea
Trifluoroethanol/NH4HCO3
0.1% SDS
0.1% RapiGest