Novel method for sample preparation for qualitative and quantitative analysis of peptidome in serum based on amino acid sequencing with a tandem mass spectrometry and novel data analysis workflow for quantitative analysis of the peptides sequenced with a tandem mass spectrometry

A mass spectrometry method for qualitative and quantitative analysis of peptidome in serum based on amino acid sequencing with a tandem mass spectrometry (MS) tool with signaling intensities measurement of peptides, characterized in that the method comprises the following steps:

• a) enrichment of peptides in the analyzed serum sample by incubation of a sample with a citrate-phosphate buffer with pH 3.1-3.6 in water with a purity≥99.9%, wherein the buffer and serum volume ratio is 29:1 and the buffer and serum are mixed at a temperature of range 4 to 8° C. to dissociate peptides from highly concentrated serum proteins;
• b) removing of proteins equal or bigger than 15 kDa from peptide enriched and simultaneously purification of peptides using hydrophilic-lipophilic balanced columns to obtain peptides smaller than 15 kDa;
• c) washing the hydrophilic-lipophilic balanced columns with high-grade water purity≥99. 9% with 0.2% formic acid (v/v) in order to remove the unbound and low-binding peptides from the columns;
• d) eluting of serum peptides bound to the hydrophilic-lipophilic balanced columns by using water/80% methanol/0.2% formic acid (v/v);
• e) filtrating of the sample through kDa molecular weight filters to collect peptides smaller than kDa;
• f) providing lyophilization of the obtained peptides by drying the eluted peptides through lyophilization for tandem mass spectrometry analysis, and then sequencing the isolated peptides based on amino acid sequences with a tandem mass spectrometry followed by intensities signaling measurements in studied serum sample in comparison with the matched control serum samples.