Thus far, one of the hurdles researchers have faced in the field of proteomics is how to achieve both sensitivity and selectivity when using a mass spectrometer to analyze a complex proteome with marked protein abundance and background interference. This poses a particular challenge in complex biological samples, such as urine or cerebrospinal fluid. The approach known as shotgun proteomics, which uses liquid chromatography and tandem mass spectrometry to analyze digested peptides, severely limits the parameters of proteomics research. Fortunately, advanced technology provides an opportunity for researchers to apply novel approaches and innovative acquisition methods to protein analysis.
In this study,1 researchers evaluated the Q-Exactive mass spectrometer (Thermo Scientific) as an alternative analytic device, which was hypothesized to enhance both selectivity and sensitivity when it comes to sample analysis. This instrument uses an orbitrap mass analyzer with a quadrupole filter for preliminary selection based on analyte mass. This setup marries the benefits of triple quadrupole instrumentation for mass filtering with the high resolution and accurate mass measurement of orbitrap mass spectrometry. A further benefit of this tool is that it allows for targeted analysis in two modes: single ion monitoring (SIM) and parallel reaction monitoring (PRM).
The SIM mode uses the quadrupole element to isolate analytes within a target range, including precursor ions. These analytes are retained in the C-trap until they are transferred to the orbitrap for MS analysis. The PRM mode isolates analytes in a similar way but then transfers them, through the C-trap, to the HCD element for fragmentation prior to analysis in the orbitrap.
To demonstrate the higher sensitivity and selectivity of this instrument when applied to proteomics research, Gallien et al. uses urine samples supplemented with digested yeast proteins and isotope-labeled peptides in varying concentrations. The researchers used a Q-Exactive mass spectrometer to analyze these samples using both SIM and PRM protocols. The data was processed using Xcalibur and/or Pinpoint for accurate data analysis. The peptide area ratios were used to define clear dilution curves for each peptide, and linear regression was performed on the curves to deduce a linearity range and estimate the quantity of endogenous peptides in the samples of urine.
Gallien et al. found that, in the SIM mode, the Q-Exactive provided researchers accurate, sensitive results in a decreased amount of time, particularly when the instrument’s multiplexing feature was used to perform up to 10 concurrent isolation cycles. In fact, the multiplexed SIM approach to analysis resulted in a 10-fold increase in sensitivity, making this mode an excellent option for quantifying peptides in complex samples. The PRM mode was also found to demonstrate marked increases in selectivity in terms of peptide fragmentation and quantification and, overall, outperformed traditional techniques. The additional selectivity of PRM mode, without a serious decrease in overall sensitivity, provides researchers a viable option for quantification in samples that generally contain background interference, such as bodily fluids.
This research has meaningful applications in the field of proteomics and in consumer-driven science. Advanced and innovative approaches allow for greater selectivity and higher sensitivity among samples that are analyzed in the lab. On the human level, the higher resolution may allow researchers to discover biomarkers and hone the methods for applying disease biomarkers to the medical consumer. In this way, technology coupled with methodological innovation yields enhanced applications in the realm of proteomic research and medical science.
1. Gallien, S., et al. (2012) ‘Targeted proteomic quantification on quadrupole-orbitrap MS‘, Molecular & Cellular Proteomics, published online September 7, 2012. doi: 10.1074/mcp.O112.019802