Focusing on the Relevant Proteome

Without enrichment of a targeted proteome, samples contain far too many proteins to be visualized in a reasonable number of analytical runs, and only the most abundant proteins are detected. Enrichment of subproteomes is necessary to reduce the sample complexity and to decrease the limit of detection. More comprehensive protein identification as well as information on subcellular localization is obtained by isolating organelles such as plasma membranes, endosomes, phagosomes, mitochondria, endoplasmic reticulum, nuclei, or Golgi apparatus from cells or tissue homogenates. Similarly, blood (plasma or serum) is enriched for lower abundance proteins by the removal of high-abundance proteins. For example, depletion of the 14 most abundant proteins using the Multiple Affinity Removal System (MARS, Agilent Technologies) removes over 96% of total protein and consequently enhances the detection of the remaining lower-abundance components [31,32]. Various depletion approaches are being used to get deeper into the plasma proteome [33]. The key criteria for implementing enrichment or depletion strategies are that they provide minimal contamination of the nontargeted proteins and are highly reproducible.

Sample Fractionation

Regardless of the origin of the purified protein samples, the complexity is typically too great—both in terms of the number of proteins and the dynamic range of concentration—for direct analysis by mass spectrometry. Reversed-phase liquid chromatography coupled to mass spectrometry (LC-MS) can reproducibly distinguish and quantify over 5000 peptides in an hour-long analytical run. A single LC- MS injection is therefore suitable for samples comprised of some 500 to 1000 proteins. Subproteome enrichment typically reduces the total number of proteins to fewer than 10,000. To characterize these complex samples more comprehensively, a fractionation step must be applied before the LC- MS. At the protein level, separations such as strong anion exchange provide effective fractionation prior to trypsin digestion and LC- MS. Alternatively, the proteins are first proteolyzed to tryptic peptides, with subsequent separation of the peptides by strong cation exchange (SCX) liquid chromatography [34] . The CellCarta work flow employs this method.

Differential Protein Expression by LC-MS

For optimal matching of peptide signals across many samples, the LC- MS platform must be as reproducible as possible (Figure 1). Technically, the requirements of stable chromatography, high resolution, and mass accuracy may be addressed by a variety of instruments. Peptide profiling takes place on mass spectrometers that are capable of providing sensitive, high-mass-accuracy measurements, such as time-of-flight or high - resolution ion- trapping instruments. Peptide sequencing is performed on quadrupole- t ime- of- flight instruments such as the QSTAR (Applied Biosystems) or the QTOF (Waters), or on an ion trap such as the LTQ-Orbitrap (ThermoFisher), which are sensitive and can provide high.quality MS/MS spectra. Regardless of the mass spectrometer type, capillary reversed-phase LC columns are coupled to these instruments via an electrospray interface. The CellCarta mass spectrometry platform utilizes capillary liquid chromatography (CapLC) and QTOF LC-MS

Human Plasma 1 Human Plasma 2 Human Plasma 3

Human Plasma 1 Human Plasma 2 Human Plasma 3

Rt (min) Rt (min) RT (min) Increasing intensity of selected ion -►

Figure 1 Consistent peptide intensity across all samples allows the detection of differentially expressed peptide ions. Shown is a partial view of peptide ion maps (as measured by LC-MS) from the plasma of three individuals. The horizontal axis is chromatographic retention time, the vertical axis is mass-to-charge ratio (m/z), and the peptide ion intensity is denoted by the size and color of the spots. The peptide ion circled shows differential expression across patients and increases in abundance from sample 1 to sample 3. (See insert for color reproduction of the figure. )

Rt (min) Rt (min) RT (min) Increasing intensity of selected ion -►

Figure 1 Consistent peptide intensity across all samples allows the detection of differentially expressed peptide ions. Shown is a partial view of peptide ion maps (as measured by LC-MS) from the plasma of three individuals. The horizontal axis is chromatographic retention time, the vertical axis is mass-to-charge ratio (m/z), and the peptide ion intensity is denoted by the size and color of the spots. The peptide ion circled shows differential expression across patients and increases in abundance from sample 1 to sample 3. (See insert for color reproduction of the figure. )

systems (Waters). The LC -MS measurements are reliable and reproducible, providing a median CV of peak intensity of 8 to 9% for peptides matched across six replicate injections of a standard sample.

To quantify the differentially expressed peptides, a suite of proprietary bioinformatics tools was developed at Caprion and implemented into CellCarta. Matching peptides across large sample sets (peak alignment) is the first step in the differential expression analysis (Figure 2). Algorithms to perform mass - to - charge ratio (m/z) and chromatographic retention-time alignment result in the confident detection of significant peptide intensity differences between samples. Peptide ions are detected and matched across all the samples in the study. Each study peptide is characterized by m/z, charge, retention time, and intensity. Bioinformatics software tools map peptides across all samples, comparing ion intensity for all reproducible peptide ions across all fractions. Those peptides that show a statistically significant differential abundance are targeted for protein identification.

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Project Management Made Easy

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