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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

Appropriate methods for the analysis of microdissected solid tumour tissues by matrix‐assisted laser desorption/ionisation‐time of flight‐mass spectrometry (MALDI‐TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing ∼...

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Published in:Proteomics (Weinheim) 2005-07, Vol.5 (10), p.2680-2688
Main Authors: Umar, Arzu, Dalebout, Johannes C. H., Timmermans, A. Mieke, Foekens, John A., Luider, Theo M.
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description Appropriate methods for the analysis of microdissected solid tumour tissues by matrix‐assisted laser desorption/ionisation‐time of flight‐mass spectrometry (MALDI‐TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing ∼200 000 cells, which were solubilised either in urea containing buffer, trifluoroethanol/NH4HCO3, 0.1% sodium dodecyl sulphate (SDS) or in 0.1% RapiGest solution, then trypsin digested and analysed by MALDI‐TOF MS. Solubilisation in 0.1% SDS resulted in detection of the highest number of sample specific peak signals. Interestingly, there was little overlap in detectable peaks using the different buffers, implying that they can be used complementarily to each other. Additionally, we fractionated tryptic digests on a monolithic high‐performance liquid chromatography column. Fractionation of tryptic digest from whole tissue sections resulted in a four‐fold increase in the total number of peaks detected. To prove this principle, we used 0.1% SDS to generate peptide patterns from 2000 microdissected tumour and stromal cells from five different breast carcinoma tumours. The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI‐TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues.
doi_str_mv 10.1002/pmic.200400128
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The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI‐TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues.</abstract><cop>Weinheim</cop><pub>WILEY-VCH Verlag</pub><pmid>15892168</pmid><doi>10.1002/pmic.200400128</doi><tpages>9</tpages></addata></record>
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subjects Analytical, structural and metabolic biochemistry
Biological and medical sciences
Breast cancer
Breast Neoplasms - chemistry
Breast Neoplasms - pathology
Carcinoma, Ductal - chemistry
Carcinoma, Ductal - pathology
Dissection
Female
Fundamental and applied biological sciences. Psychology
Gynecology. Andrology. Obstetrics
Humans
Laser microdissection and pressure catapulting
Mammary gland diseases
Matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry
Medical sciences
Miscellaneous
Neoplasm Proteins - chemistry
Neoplasm Proteins - isolation & purification
Peptide profiling
Peptides - chemistry
Peptides - isolation & purification
Protein Array Analysis
Proteins
Sensitivity and Specificity
Solubility
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tumors
title 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
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