Figure 2.

Workflow for identification, verification and application of biomarkers in clinical diagnostics. Stable isotope labeling with amino acids in cell culture (SILAC), difference gel electrophoresis (DIGE) and isobaric tags for relative and absolute quantification (iTRAQ) are all powerful tools for finding differences in protein production in separate samples. A marker is added to the samples during either the experiment or the preparation for analysis; the samples are then analyzed together and in the resulting data can be told apart on account of the different markers. The aim is to find proteins that significantly differ in expression between the samples. For further validation, reverse-phase protein array (RPPA), and especially multiple reaction monitoring (MRM), are highly sensitive methods that can detect subtle differences in production of proteins identified as potential biomarkers. RPPA is an antibody-based assay that detects and quantifies protein production. MRM allows detection and absolute quantification of protein based on internal standard peptides. A suitable peptide, fulfilling the criteria to enable optimal analysis, is chosen from within the target protein and then produced with a heavy isotope amino acid incorporated. This synthetic peptide is added in known amounts to the sample. In the triple quadruple instrument (QQQ), the peptides of interest are selected (Q1), fragmented (Q2) and the resulting target peptide ions selected (Q3) for detection. As the amount of standard peptide added to the sample is known, peak comparison allows calculation of the amount of the target protein present in the sample. To apply identified and validated biomarkers in clinical diagnostics, the analytical method must be highly reproducible. Flow cytometry is a well-established method of analyzing hematological samples. With the application of mass spectrometry detection after flow cytometry selection (ICPTOF-MS), problems with multiplexing are overcome, and this method enables detection of up to 20 biomarker proteins. The nanofluidic proteomic assay (NIA) method allows quantitative detection of protein production in very limited material. The proteins are separated according to isoelectric point inside capillary glass tubes before immobilization and antibody detection.