The dynamics of time, space, and concentration, as well as variability due to modifications and mutations of proteins make proteomics considerably more complex than genomics. Therefore several complementary mass spectrometry (MS) approaches are required to generate a complete picture of what a protein is, where it might be located, and how it is modified. For proteomics researchers looking to do more than identify and quantify an individual protein, or who want to elucidate their understanding of a single cellular compound, combining MS approaches offers advantages over a single MS technique.
We outline three techniques, and the MS systems utilized for them, that can be used individually or as a complementary approach. Because each of these techniques sheds slightly different insight into and information about the protein, collectively they help establish in-depth knowledge, not mere data. Choosing an MS vendor that supports all the necessary tools, including bioinformatics, helps achieve multidimensional analysis.
- Bottom-up analysis: Protein characterization by comprehensive post-translational modification (PTM) analysis, including glycosylation. One method for protein analysis is to digest larger proteins into smaller, more manageable, fragments. Techniques to consider include: Ion trap systems that combine collision-induced dissociation (CID) MSn and electron transfer dissociation (ETD) fragmentations to fully describe and locate classic or unusual PTMs; MALDI-TOF/TOF systems to screen huge datasets and search for specific fragmentation pattern, or allow both peptide and glycan information from a one-shot analysis of a glycopeptide; and highly accurate ultra-high resolution quadrupole time-of-flight (UHR-Q-TOF) MS/MS data to offer true de novo sequencing and very high mass accuracies that provide more information when highly complex mixtures are to be analyzed. It should be noted that bottom-up requires support by advanced bioinformatics capabilities and vendor software with the potential of merging information from different origins.
- Top-down approach: Offers the advantage of proteoforms distinction and quantitation, to complement the more classical and bottom-up approaches: Mixture of proteins with different post-translational modification patterns, or mixtures of protein fragments issued from proteolysis events can then be deciphered, providing the biologist with precious information on regulation mechanisms. Top-down approaches use intact proteins, instead of the digested protein that is analyzed by its digested fragments. Here, the Q-TOF will excel in measuring the accurate mass of different proteoforms separated by LC, in quantifying them and in generating tags to enable sequence identification. Longer tags can be generated by ETD ion trap, Q-TOF and Fourier transform ion cyclotron resonance (FT-ICR) from mixtures. However, if the protein can be isolated and concentrated to picomole levels, a MALDI-TOF/TOF system will show an unparalleled capability to generate the longest sequence tags within seconds, enabling, for instance, rapid and accurate QC of recombinant proteins and biopharmaceuticals.
- MALDI imaging for on-tissue proteomics: Cellular location of a given proteoform completes the picture (literally). The latest techniques enable mapping 3D distribution of molecular species or quantification. The imaging approaches, whether top-down or bottom-up, are currently restricted to the most abundant species. However, the physiologic distribution of the proteoforms gives precious information to the biologist. While measuring intact protein distribution, this information can reveal the action of proteases or other modifying enzymes on different cellular locations, for example, drug-resistant vs. drug-sensitive cancer cells. This information is not accessible via the classic or shot-gun bottom-up approaches, where all proteoforms issued from the same gene expression are mixed and digested together.
- Combining MS techniques yields the most information: Using a multidimensional approach yields significantly more information than “one size fits all” mass spectrometry. No single technique or instrument can deliver a true picture of the proteome, complete with biological context and certainty in data quality. Combining bottom-up, top-down PTM analysis with glycosylation assignments and MALDI molecular imaging of intact proteins cuts through the complexity to deliver a comprehensive view. Coupled with a state-of-the-art bioinformatics solution, researchers using multiple MS approaches gain knowledge from the vast collection of data generated. Experienced MS vendors with a full range of solutions and integrated software can tailor multidimensional approaches that best meet the specific needs of researchers.