The application of mass-spectrometry-based protein biomarker discovery to theragnostics

Abstract

Over the last decade rapid developments in mass spectrometry have allowed the identification of multiple proteins in complex biological samples. This proteomic approach has been applied to biomarker discovery in the context of clinical pharmacology (the combination of biomarker and drug now being termed 'theragnostics'). In this review we provide a roadmap for early protein biomarker discovery studies, focusing on some key questions that regularly confront researchers.

Figure 1

Schematic representation of a liquid chromatography–tandem mass spectrometry (LC-MS/MS) instrument consisting of five parts; the LC column, the electrospray apparatus, the primary MS, the collision cell and the secondary MS. A sample loaded on the LC column is eluted in an increasingly concentrated acetonitrile solution and the eluate fed into the electrospray apparatus. The eluate is ejected from an electrically charged glass needle as a stream of ions that are taken in by the first mass analyser (quadrupole). This mass analyser operates in two modes. In scanning mode it surveys the m/z ratios for all the ions entering the mass analyser. When an interesting ion is identified this mass analyser switches to an ion selection mode and feeds this ion into the collision cell, where it is fragmented. The fragmentation products then enter the second mass analyser, where their m/z ratios are measured. The combination of the parent ion m/z ratio and the fragmentation ion m/z ratios is highly specific and enables the parent peptide to be identified with high confidence

Figure 3

Flowcharts representing the main approaches for quantitative mass spectrometry. Vertical arrows represent sample processing steps. The introduction of label and the point at which samples are combined are labelled for each approach. Black and white shapes indicate discrete experimental conditions. Grey shapes represent the combined sample

Figure 2

Peptides are eluted from the liquid chromatography column over a defined period of time. Integrating the mass spectra over time, represented by the highlighted section of the chromatogram (top panel), gives the total ion count for a species (highlighted region of centre panel). This can then be compared between experimental conditions. The identity of the species being quantified can be deduced by examining the mass spectrometry (MS)/MS spectra (bottom panel)

Figure 4

Schematic representation of isobaric tag for relative and absolute quantification (iTRAQ) quantitative mass spectrometry (MS). Protein samples from four experimental conditions are digested and labelled with isobaric tags. The mass of the tag is kept constant, represented by a constant length, by varying the mass of the balance group to compensate for the mass of the reporter group. This means that for the first MS analysis peptides from the four experimental conditions cannot be differentiated. Following ion fragmentation in the collision cell the reporter group is liberated and can be detected on the second MS analysis. The peaks for the reporter groups (mass 114.1–117.1) are present in a region of the mass spectra with generally low background. The diagram shown here suggests a relative abundance for this peptide of 3:2:4:1 for the four experimental conditions