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Exploring the Fourth Dimension of Proteome Analysis

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Just a few years ago, it was a successful feat for a proteomics researcher to identify the proteins, peptides and post-translation modifications in a single or small number of samples. Fast-forward ten years, and the field has changed dramatically.

Now, scientists want to conduct analyses on a larger scale, with sample numbers extending beyond the hundreds into the thousands. This analysis must be of a high-quality, with high-sensitivity and specificity, yet efficient – particularly when considering the applications of proteomics in the personalized medicine and biomarker detection spaces.

And so, mass spectrometry (MS) instrumentation continues to evolve on an extraordinary scale. Whilst liquid chromatography-mass spectrometry (LC-MS) remains the "gold standard" technology available for the high-throughput characterization of proteins, scientists in the field are continually pursuing methods to gain a deeper analysis of their samples.1

Recent developments in MS technology have enabled a fourth dimension of analysis: ion mobility separation. The term "4D proteomics" has been created to describe analysis that incorporates all four dimensions.

Adding the fourth dimension

In a classic bottom up LC-MS experiment, peptides are digested as they elute from the LC column. Here, the time taken for a solute to pass through the column after being injected is known as the retention time (RT). The determined RT is one dimension of analysis. The mass obtained for the peptide is the second, and the intensity of the of the peaks on the spectra is the third.

The fourth dimension of analysis is achieved using ion mobility separation (IMS), an analytical chemistry method that can be coupled with a standard LC-MS/MS workflow to form ion-mobility spectrometry–mass spectrometry (IMS-MS).

Gary Kruppa, PhD, Vice President of Proteomics, Bruker Daltonics Inc., told us: "The invention of trapped ion mobility spectrometry (TIMS) by Bruker has made the routine use of ion mobility in proteomics possible." 

"As multiple peptides co-elute off the nano-LC column, their unique collision cross sections (CCS) allows for further gas phase separation in the TIMS cell, allowing for more peptides to be identified. This gas phase separation as a result of TIMS is the fourth dimension in addition to retention time, mass-to-charge, and intensity, and termed as 4D matching," he adds.

4D matching enables researchers to identify proteins that are of lower abundance, such as signaling proteins, with high sensitivity. The CCS value can be utilized as an additional identification point in data dependent analysis. In data independent analysis, the CCS value works as a unique signature to align features.

Recently, we interviewed two researchers at the forefront of the proteomics field, 
Professor Jürgen Cox, Group Leader at the Max Plant Institute of Biochemistry and Dr Roman Fischer, head of the Discovery Proteomics Facility of the Target Discovery Institute to learn more about how 4D proteomics approaches are enhancing their research.