Classic proteomic workflows analyze tryptic peptides, which generally weigh less than 3000 Da, using a "bottom up" approach.
Volume 30 Number 2
Classic proteomic workflows analyze tryptic peptides, which generally weigh less than 3000 Da, using a "bottom up" approach. The importance of multiple and coordinated post-translational modifications compels the analysis of longer peptides (using "middle out" analysis) and intact proteins (using "top down" analysis). Professor Catherine Fenselau of the Department of Chemistry and Biochemistry at the University of Maryland is a leading researcher in this field, and has won numerous awards for her work, the most recent of which is the 2014 Eastern Analytical Symposium Award for Outstanding Achievements in Mass Spectrometry. She recently spoke to us about her research with mass spectrometry–based analyses of proteins associated with cells known to be an obstacle to anticancer immunotherapies.
A recent article of yours (1) describes the bottom-up analysis of the proteome of exosomes (vesicles that exchange genetic material between cells) shed by myeloid-derived suppressor cells (MDSC), which are present in most cancer patients and inhibit natural antitumor immunity. How did the collaboration for this study come about?
Fenselau: Professor Suzanne Ostrand-Rosenberg and I are friends from the decade I spent at the University of Maryland, Baltimore County (UMBC), where she still works. The interests and skills of our laboratories are highly complementary, and together our scientific reach is much broader than either alone. We started studying plasma membrane proteins (my specialty) from MDSC (Sue's specialty) about six years ago, and recently, in a high-energy conversation, we activated our mutual interest in exosomes and their protein content.
What was the most challenging aspect of the mass spectrometry (MS) analysis in this study?
Fenselau: As is often the case, the biggest challenges are obtaining enough sample and a pure sample of proteins from the exosomes.
How did your group use bioinformatics to characterize the proteome of these exosomes?
Fenselau: One of the coauthors in this work, Professor Nathan Edwards (Georgetown University), is a leader in protein bioinformatics. We used the powerful meta-search program he has written - PepArML - that combines results from seven open source search engines to identify our proteins with high reliability. We searched our mass spectra against the mouse protein database from UniProt Knowledgebase, the global repository for protein sequences and other information.
A second article from your group describes the top-down analysis of low-mass proteins in the same type of exosomes (2). What size proteins are being examined in this study and why are they important?
Fenselau: We have developed our own workflow to obtain top-down analyses of proteins below about 25,000 Da. We are limited in mass range by the capabilities of our mass spectrometer; however, this mass range includes nearly half of the proteins predicted to be translated from the human genome.
In our study of the proteins in exosomes, we were surprised to find that more than half are histones. Histones are usually associated with chromatin, which is not present in exosomes. We have also shown that MDSC exosomes carry a wide range of RNAs, and we suggest that the histones serve as chaperones for these.
Why did you decide to apply a top-down approach to this particular analysis? How does the top-down analysis you used differ from standard approaches with respect to the sample preparation and MS method used?
Fenselau: We knew from our bottom-up investigation that many of the proteins in our sample are histones, well known to be extensively and variably modified. We wanted to profile the modifications in our histones to help us understand their role in MDSC exosomes. Top-down analysis is the best strategy to assess multiple modifications on individual proteins.
In a top-down workflow we prepare protein samples without proteolysis. This means we need a high performance liquid chromatography (HPLC) column and gradient suitable to fractionate proteins; we must be able to activate heavier (protein) ions to produce informative fragmentation; sufficient mass resolution is required to assign charge states of both precursor and product ions; specialized search programs are needed.
Ubiquitin is a small (8.5 kDa) regulatory protein that is associated with almost all eukaryotic organism tissues, and post-translational modification of proteins with ubiquitin, or ubiquitination, codes proteins for regulation of cell differentiation, apoptosis, endocytosis, and other functions. Your group used a middle-out MS-based strategy with LC–MS-MS to analyze lysine-linked ubiquitin dimers (3). How are these dimers being used to study the ubiquitin code?
Fenselau: The ubiquitin code is expected to correlate patterns of ubiquitination with the functional fates conveyed to protein substrates by conjugation. The code has been only partly deciphered because no suitable analytical method is available. In addition to monoubiquitin, a large variety of polyubiquitin chains can be attached to target proteins, which vary in length and branching patterns. We are developing methods to characterize these complex critical modifications, and we started with the set of isomeric dimers described in reference 3. Presently we are working with trimers and more complex polyubiquitins, which are synthesized and made available by Professor David Fushman (University of Maryland) and his team.
What are the advantages of using tandem MS following reversed-phase liquid chromatography to characterize these dimers?
Fenselau: Tandem mass spectrometry provides the best combination of sensitivity and specific structural information for any bioorganic sample. This, and its suitability for database searching, has made it the method of choice for proteomic research. LC–MS-MS brings the same strengths to analysis of these branched polypeptides.
Why is a middle-out approach a better choice in this case than a top-down or bottom-up approach?
Fenselau: A bottom-up approach with complete tryptic cleavage will not characterize sequential branch points in polyubiquitins. Top-down is not readily applicable to heavier polyubiquitins because their mass is too high for the capabilities of current mass spectrometers. The middle-out approach reduces the mass by trimming the branches, and allows the trunk or backbone with sequential branch points to be recovered for easier analysis.
An MS-based bottom-up proteomics approach was used by your group in an effort to identify ubiquitinated tryptic peptides carried by exosomes derived from myeloid-derived suppressor cells and to identify sites of ubiquitination on these peptides (4). What MS technique was chosen for this study and why?
Fenselau: This exploratory study was the first to identify ubiquitin-conjugated proteins in exosomes. We used a conventional bottom-up workflow because it offered the greatest sensitivity. We are combining our protein identifications with western blotting to recognize ubiquitinated proteins present in sufficient amounts to allow us to take the next step - complete elucidation of polyubiquitin modifications.
You noted in your presentation at the Eastern Analytical Symposium that there is a need for faster instrumentation and better mass resolution and accuracy. How would those improvements benefit your research?
Fenselau: For various reasons, top-down analyses in current instrumentation require longer times for scanning and activation. This means that fewer proteins can be analyzed in a complex mixture during each HPLC fractionation. In addition, many instruments have reduced resolution and mass accuracy at the higher masses to which we aspire in top-down work. We expect that continuing development of the capabilities of both orbital ion trap and time-of-flight mass spectrometers will eventually extend top-down protein analysis to heavier proteins and complex mixtures.
What are the next steps in your research?
Fenselau: Our overall objectives are to develop a reliable method to characterize the structures of multiply branched polyubiquitin protein modifications and to demonstrate this approach in exploring the cargo of immunosuppressive MDSC exosomes. We have identified targets for the latter effort and initiated structure elucidation as mentioned above. Among multiple efforts to advance the analysis of these branched peptides and proteins, we are working on novel fractionation methods, enhanced MS-MS fragmentation methods, and algorithms for automated interpretation of tandem mass spectra.
(1) C.M. Burke, W. Choksawangkarn, N. Edwards, S. Ostrand-Rosenberg, and C. Fenselau, J. Prot. Research13, 836–843 (2014).
(2) L. Geis-Asteggiante, A. Dhabaria, N. Edwards, S. Ostrand-Rosenberg, and C. Fenselau, Int. J. Mass Spectrom. DOI: 10.1016/j.ijms.2014.08.035 (2014).
(3) A.E. Lee, C. Castaneda, Y. Wang, D. Fushman, and C. Fenselau, J. Mass Spectrom. DOI 10.1002/jms.3458 (2014).
(4) M. Burke, M. Oei, N. Edwards, S. Ostrand-Rosenberg, and C. Fenselau, J. of Prot. Research DOI 10.1021/pr500854x (2014).
This interview has been edited for length and clarity. For more interviews on spectroscopy-related techniques, please visit spectroscopyonline.com/spectroscopy-interviews