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- Bllaci, Loreta, et al.
(författare)
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Fast Surface Acoustic Wave-Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Cell Response from Islets of Langerhans.
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 85:5, s. 2623-2629
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Tidskriftsartikel (refereegranskat)abstract
- A desire for higher speed and performance in molecular profiling analysis at a reduced cost is driving a trend in miniaturization and simplification of procedures. Here we report the use of a surface acoustic wave (SAW) atomizer for fast sample handling in matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) peptide and protein profiling of Islets of Langerhans, for future type 2 diabetes (T2D) studies. Here the SAW atomizer was used for ultrasound (acoustic) extraction of insulin and other peptide hormones released from freshly prepared islets, stimulated directly on a membrane. A high energy propagating SAW atomizes the membrane-bound liquid into approximately 2 μm diameter droplets, rich in cell-released molecules. Besides acting as a sample carrier, the membrane provides a purification step by entrapping cell clusters and other impurities within its fibers. A new SAW-based sample-matrix deposition method for MALDI MS was developed and characterized by a strong insulin signal, and a limit of detection (LOD) lower than 100 amol was achieved. Our results support previous work reporting the SAW atomizer as a fast and inexpensive tool for ultrasound, membrane-based sample extraction. When interfaced with MALDI MS, the SAW atomizer constitutes a valuable tool for rapid cell studies. Other biomedical applications of SAW-MALDI MS are currently being developed, aiming at fast profiling of biofluids. The membrane sampling is a simplistic and noninvasive collection method of limited volume biofluids such as the gingival fluid and the tearfilm.
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| 2. |
- Bllaci, Loreta, et al.
(författare)
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Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins
- 2017
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:21, s. 11332-11340
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Tidskriftsartikel (refereegranskat)abstract
- Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific, and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY-imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP- and TiO2-based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO2 combination as compared to TiO2 alone. pY constituted up to 8% of the pY-MIP-TiO2-enriched phosphopeptide fractions. The pY-MIP-TiO2 and the TiO2 protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842, respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO2-enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic, and almost exclusively monophosphorylated, as compared to TiO2 alone. These unique properties render the pY-MIP-based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.
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