Differential peptide display

After separation on a RP-HPLC column employing an ACN-gradient (4 to 40%) and collection of 96 fractions, a 15 mL equivalent of plasma was subjected to MALDI-TOF MS (for details see [5]) (Voyager DE STR, Applied Biosystems). The MALDI matrix consisted ofalpha-cyano-4-hydroxycinnamic acid (matrix) and 6-desoxy-l-galactose (co-matrix) in ACN containing 0.1% TFA. Data were analyzed, including peak recognition and visualization using the software package Spectromania®. Quantification of mass spectrometric signals was performed after baseline correction by integrating absolute signal intensities in 1 Da bins. Using the software, the mass spectrum of each fraction was transformed to a virtual lane. The molecular weight of each peptide is indicated by its position within the virtual lane, whereas the MALDI-signal intensity for each peptide is depicted by the color intensity of the corresponding bar. The converted mass spectra of all 96 fractions were combined resulting in a 2-D display of peptide masses termed peptide map display. Detection of differentially expressed peptides was achieved by calculation of subtractive peptide maps displays or correlation analysis (differential peptide display) by referring to mass spectrometric data [3-5].

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