INVESTIGADORES
MORENO Silvia Margarita
congresos y reuniones científicas
Título:
Improving recovery of manual trypsin in-gel digestion for high throughput protein
Autor/es:
M.R. GIROTTI, D.CAPORALETTI, S.MORENO Y A. LLERA
Lugar:
Pilar, Buenos Aires
Reunión:
Congreso; Primer Congreso Anual Iberoamericano de Proteómica; 2007
Institución organizadora:
LAHUPO
Resumen:
Nowadays mass spectrometry-based proteomics is an essential tool for molecular, cellular and  system biology. As a result of its mass accuracy, high resolution and sensitivity, MALDI–TOF (matrix-assisted laser desorption/ionization-time of flight) is used to identify proteins by peptide-mass fingerprinting combined with peptide sequencing matched against databases. In this work we aimed to improve the recovery of manual in-gel digested spots to enhance sensitivity of the technique for the identification of proteins in a high-throughput format. We used plant (FBPase from Brassica napus) and animal proteins (actin from human and Mus musculus) as controls. We excised gel spots from one or two-dimensional gels either stained with SYPRO Ruby or silver nitrate. In order to set up a protocol that can be applied to isolated proteins coming from different sources, we tested several conditions. We optimised the size of gel spot to be processed and the length of the tryptic incubation. We also tested various matrix concentrations and the matrix:sample proportion to choose the one with best desorption yields. Our results also show that pretreatment of plastic and glass materials with a mixture of acetonitrile/TFA/water, the avoidance of sample vacuum concentration to dryness and a final centrifugation step before sample deposition in target were helpful to recover a suitable amount of peptides without undesirable polymer contaminations. Peptide digests were analyzed by a MALDI TOF-TOF instrument present in CEQUIBIEM facility. The sequence coverages and average peak intensities obtained from this analysis were compared. We could identify the control proteins extracted in optimized conditions with high MASCOT scores. The information obtained at the present work will be applied to improve the service in the recently available protein identification facility.