Much like the latest Intel chip in your computer, which is considerably better, faster, and cheaper than the previous version but largely unheralded, no one gets excited about mass spec advances anymore, claims John Yates, professor, department of chemical physiology, The Scripps Research Institute. “Although the state-of the-art is impressive and considerably accelerates the field of proteomics in its traditional applications and new ones, the advances have become so routine they’ve become ho-hum.”
However routine should not be misconstrued as irrelevant or ineffective. Proteomics technology is advancing, and mass spec remains at the core of many scientific discoveries
“Mass spec is now considered the best way for identifying and quantifying proteins in single cells—unmatched by the other current methods such as Western blots, CyTOF, and RT-PCR,” remarks Daniel Lopez-Ferrer, senior strategic marketing specialist proteomics at Thermo Fisher Scientific.
Traditional proteomics
The bread-and-butter application of mass spec is the “bottom up” approach of interrogating which proteins are associated with a “pulled down” (usually immunoprecipitated) protein of interest. The proteins are then digested into peptides by an enzyme, typically trypsin, and then loaded into the mass spectrometer that separates the peptides based on mass and charge. Based on calculations of atoms’ mass and charges, the read-out can be used to sequence and quantify the proteins that were in the sample. This can be considered proteomics because it involves getting a snapshot of proteins’ relationships to one another.
New technology has expanded the types of proteomic analysis that can be accomplished.
Devising an “interaction map” between proteins is a mainstay of proteomics and basic science research. And even here, new technology has expanded the types of proteomic analysis that can be accomplished. Some of the innovations are outside the instrumentation. For example, Promega makes enzymes that digest the proteins in different ways, work in more natural conditions, different pHs, or cleave various types of modifications. “Now that it’s easier for post-translational modifications to be analyzed, comparing proteins from different species or conditions to look at what happens to a phosphorylation or glycosylation event is becoming more common. Mass spec can also handle more complex and ‘natural’ samples such as blood and cerebral fluid as opposed to only tissue culture; this enables results that are more likely to reflect real-life situations,” explained Gary Kobs, senior global product manager from Promega.
Another major mass spec advance that has taken place in the past five years is “top down” proteomics where instead of digesting the protein sample into peptides before it is loaded onto the mass spec, the “whole protein” is interrogated. This enables various forms of protein in the sample to be identified. Neil Kelleher, professor of chemistry, Northwestern University, one of the pioneers of the top down mass spec proteomics approach, says that mass spectrometers need to have good fragmentation capabilities built-in for this type of proteomics.
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Better dissociation techniques within the instruments have made recent progress possible. Applications in basic research have been in the field of epigenetics for analyzing post-translational modifications such as histones. Top down approaches are also proving very useful in the area of clinical diagnostics where, for example, there may be different forms of a hormone that need to be identified.
Biomarker research booming
Biomarker research is another area that has significantly benefitted from mass spec advances. Using mass spec to identify and quantify the proteins in a diseased cell versus non-diseased cell is much easier now. As Christine Hunter, director of omics applications, SCIEX, explains, targeted mass spec for targeted proteomics can now be accomplished in a more high-throughput fashion because of hybrid mass specs that allow greater mass accuracy as well as better sensitivity. Instruments such as a hybrid between a quadrupole and linear ion trap are such an example. Newer products also contain innovations on the front-end (sampling) as well as back-end (software). “These newest mass spec offerings are helping biomarker research by enabling larger scale ‘industrialized proteomics’—more samples per day can be processed because of automation of sample prep and faster downstream work via cloud computing,” she adds.
Greater automation is helping biomarker research flourish because larger studies can be carried-out. Hunter cites an example of how a study involving a cohort of 400 tissue samples at a rate of 1hour/sample can now be completed in a few weeks as opposed to months before.
Biotherapeutics quality assurance
If you define proteomics in its more general sense of “studying how proteins are modified and changed in various systems” there is a whole new field emerging of applying mass spec to the characterization of therapeutic biological drug candidates. Michelle English, senior scientist, analytic R&D department, mass spec and biophysical characterization, Pfizer, describes her line of work as one that is increasingly becoming more focused on mass spec applications in proteomics. Mass spec analysis has become a mainstay for her in investigating whether biologics, or antibody-based drugs in development are the composition they are supposed to be, without any new protein modifications or “contaminating” proteins introduced during their production.
“We mainly use mass spec to check for sequence fidelity—did a mutation occur that is changing the sequence of the antibody? But there are newer applications for mass spec as well thanks to advances in instrumentation that provide good resolution, which enables detection of less abundant host cell proteins or sequence variants,” English explains.
Applying mass spec to pharmacokinetic associations to characterize biologic drugs is a growing field as well, explains Kobs. “ASMS in June was filled with posters and talks about antibodies and antibody-drug conjugates. Our customer base in the mass spec arena is now a majority of pharmaceutical companies and CROs.” Last month, Promega started to offer a low pH-functioning trypsin so that the mass spec sample prep of antibodies can be performed at a lower pH in order to avoid artificial post-translational modifications being added to them and introducing artefacts.
Biomarker Detection from Tissue Sections
MALDI Tissuetyping of a lung cancer sample. Ion images of cytoplasmic actin (A) and collagen alpha-1 (1) chain precursor (B) show their prominence in tumour and stroma regions, respectively. Merge of the two (C); actin in red and collagen in green) demonstrates clear delineation between tumour and stroma. (D) Staining of the section post measurement confirms the histological annotations. Scale bar indicates 100 µm.
MALDI imaging helps pathology researchers gain new insights in tissue. Unlike other techniques it does not require target specific reagents. It allows the direct visualization of molecules such as proteins, peptides, or glycans in their histological context. Tissuetyping is a nondestructive technique that allows the histological staining of the tissue and microscopic analysis after MALDI measurement. The molecular distributions can be directly overlaid with the microscopic data.
Bruker’s MALDI Tissuetyper can support tissue research in different ways. The data can be used to look for tissue-specific marker molecules, but since the distributions of many molecules are measured simultaneously one can also use these expression patterns for more sophisticated analyses. For instance, it is possible to use these patterns to train classifiers for specific tissue states in an attempt to objectively classify unknown tissue states. Similarly, tissue segmentation based on molecular profiles allows a detailed ab-initio look into tumor heterogeneity.
Image: Brucker