Caprion's technology platform covers three main components: sample preparation, mass spectrometry analysis and bioinformatics.
Successful proteomic studies require careful sample preparation in order to yield relevant and reproducible results. Typically, samples are subjected to some form of fractionation which focuses in on the proteome that is most relevant for the particular study. For biological fluids such as plasma, serum or CSF, Caprion depletes the samples of their most abundant proteins (for example immunoglobulins and albumin) that would otherwise mask lower abundant proteins of interest. A well-established immunoaffinity approach is utilized. For tissue or cells, Caprion has developed protocols to enrich for the relevant fraction such as plasma membrane or proteins in the secretory pathway. Samples are tracked at all time and strict Standard Operating Procedures are applied to minimize variability. Once protein samples are prepared, they are typically subjected to trypsin digestion prior to analysis by mass spectrometry.
Mass Spectrometry Analysis
Caprion uses two mass spectrometry-based approaches for discovery and targeted protein measurements, respectively.
Using its proprietary ProteoCarta™ (formerly CellCarta®) platform, Caprion performs discovery unbiased, hypothesis-free studies whereby the proteome of a large numbers of samples is profiled in order to identify proteins whose abundance differs between study groups or cohorts. Endogenous peptides obtained from digestion with trypsin are first analyzed in scanning mode by mass spectrometry coupled to reverse phase chromatography (LC-MS). As the peptides elute from the column their mass-to-charge ratio (m/z) and corresponding signal intensities are determined, resulting in a three-dimensional map whereby each peptide is assigned a retention time, an m/z value and an intensity value.
Next, these maps, which track up to 15,000 peptides per sample, are matched across samples and statistical analyses are performed to identify which peptides differ between study groups of interest.
In parallel to the profiling step, a subset of samples is subjected to tandem mass spectrometry (LC-MS/MS) whereby peptides are fragmented to generate spectra that are matched to predicted spectra from well-characterized databases. In this way, up to thousands of proteins, including those that are differentially expressed between samples, are identified.
Multiple Reaction Monitoring Assays
Caprion also develops and deploys targeted multiplexed assays to measure specific proteins of interest, using multiple reaction monitoring (MRM) technology. Protein biomarkers can be selected from genomics, literature, or discovery efforts. MRM uses triple quadrupole mass spectrometers coupled to liquid chromatography. In the first quadrupole, a specific peptide that corresponds to a protein of interest is selected. The peptide is then fragmented in the second quadrupole and a filter is applied to allow a specific fragment, also referred to as a transition, to enter into the third quadrupole where its intensity is measured. Typically, two peptides per protein are monitored and two transitions per peptide are measured. Because of the double selection approach, which improves signal-to-noise and reduces interference, and multiple measurements per protein, MRM is one of the most specific assays for protein measurement. In addition, current instrumentation allows for the measurement of hundreds of proteins in a single sample, making MRM an ideal assay to verify and validate candidate biomarkers or perform high-throughput measurements of a panel of markers.
Principle of MRM Measurements
MRM can also be coupled to immunoaffinity enrichment of target proteins to further increase sensitivity. Such anti-body-assisted MRM assays are highly linear and when applied against plasma proteins, typically show lower limits of quantification in the low nanogram per ml range.
Antibodies that display cross-reactivity ca be used in such assays since the specificity is brought about by the MRM assay component.
Leveraging its multidisciplinary Bioinformatics team, Caprion has developed a robust and integrated suite of software tools and databases to efficiently process and analyze mass spectrometry data. Embedded in its proprietary informatics pipeline are the Elucidator software for peptide quantification and Mascot software (Matrix Sciences) for protein identification. Caprion has also developed a number of software tools to automate such steps as peptide selection for targeted studies, MRM assay development and data mining from discovery studies. The use of IPA (Ingenuity Systems) and proprietary software facilitates the integration of annotation and literature data with the complex datasets generated by Caprion, allowing better interpretation and visualization of the results in their biological context. All statistical analyses performed by Caprion are carried out by expert PhD level biostatisticians, experienced in a wide variety of statistical methods. Biostatisticians are involved in several steps of the research process, from study design, to data analysis and interpretation to insure the validity and significance of the findings.