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This technique combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. The peptides will be separated on a nano-ACQUITY Ultra Performance UPLC™, (Waters MS technologies, Manchester, UK), using a reverse phase column. Peptide analysis is performed with MSE on the nano-UPLC™ Synapt G2 (Waters MS technologies, Manchester, UK). This instrument allows for increased detection performance of low abundance proteins and peptides, as well as simultaneous identification and sequencing of detected peptides. The mass spectrometry method is a known technique used for Label-free proteomics.
Identification and quantification is accomplished using TransOmics (Nonlinear dynamics, UK). Databases Uniprot and SwissProt will be used for identification and bioinformatics search.
SIMCA-P+, v 13.0 (Umetrics AB, Umeå, Sweden) is used for multivariate statistics, including orthogonal projection to latent structures – discriminant analysis (OPLS-DA).