Protein identification presents numerous challenges to life science researchers. Proteins can be difficult to detect, very large or very small, and have perplexing chemical properties. The dynamic range of abundance of proteins found in nature is perhaps the greatest challenge, and means that any comprehensive screening program must incorporate multiple stages and multiple technologies in order to capture all significant proteins.
Pharma industry applications for protein identification technologies include biomarker discovery and validation, protein target discovery and validation, protein-protein interactions, antibodies, analysis of gene knockout or gene silencing experiments, and companion diagnostic development for pipeline drugs. There is a need for more specific proteomics identification tools in today’s drug discovery—for example, researchers often need to distinguish isomers or post-translational modifications.
Much new innovation in proteomics centers on improvements and refinements of mass spec. Novel approaches to complementary proteomics technologies such as the immunoassay and light scattering technology provide additional options for identifying and characterizing proteins.
Mass spec like never before
“The TripleTOF 5600 system is being deployed in big protein biomarker studies where the power of the TripleTOF is needed to dig deeper into a sample, especially to identify low abundant proteins,” says Dominic Gostick, director of academic research at AB SCIEX.
Gostick adds that the 5800 system is being used for routine protein identification, while the 5600 can be used for identification as well as quantification in advanced workflows.
The Waters Corporation (Milford, Mass.) approach to mass spec incorporates nano-scale UPLC separation using a data-independent acquisition strategy. It differs from many other approaches in that it is not necessary to select peptide ions in real time to fragment them. Data can be acquired on all detectable species, all the time, by cycling between two modes alternating low and high collision energies.
“This allows both peptide accurate mass information and fragment ion accurate mass information to be generated for database searching,” says Waters spokesman James Langridge. “This approach results in extremely high-duty cycle, sensitivity, and dynamic range, allowing comprehensive measurements to be made in either simple or complex protein mixtures.”
An extension on the UPLC system adds HDMS (high-definition mass spectrometry) functionality to the system. “The HDMS-E approach uses ion mobility as an additional gas phase separation technique,” Langridge explains. “This gives a significant increase in peak capacity, separation efficiency, and as such allows us to probe complex mixtures in a robust and reliable fashion like never before.”
Ion mobility separation permits the detection of both high- and low-abundance peptides. By using the accurate mass of the precursor peptides and their fragments, it is possible to identify proteins with very high sensitivity and selectivity.
Post-translational modifications demystified
Electron transfer dissociation (ETD) is a new type of fragmentation technique for protein identification that complements other methods such as collisionally-induced fragmentation and higher energy collisional dissociation. ETD is able to fragment and sequence highly modified peptides without loss of the modification. The preservation of the post-translational modification enables precise determination of the site of that modification, for example, mapping the exact location of multiple phosphorylations on a histone.
ETD can also sequence larger proteins, often for more than 40 amino acids at each terminus. In combination with other types of fragmentation technologies, ETD provides very comprehensive coverage of the protein.
Common applications for ETD include its use on a hybrid linear ion trap instrument to identify highly modified histones or other hyperphosphorylated proteins and acetylation of metabolic enzymes. “ETD really enables biologists today to study protein isoforms eventually identifying the biologically active protein isoform that is responsible for regulatory events in the cell,” says Andreas Huhmer, PhD, director of proteomics marketing for Thermo Fisher Scientific (Pittsburgh, Pa.). “This is a dramatic shift from the major objectives of proteomics a few years ago, when the focus was on principle discovery of gene products.”
In the field of drug discovery, interest in epigenetic drug targets is heating up. There is a growing need to identify epigenetic targets and develop inhibitors that are specific to post-translationally modified proteins. Merck researchers used ETD to study isoform-specific inhibitors of class 1 and class 2 histone deacetylase enzymes. The group profiled histone-enriched cellular fractions treated with various inhibitors and analyzed them using differential mass spectrometry using EDT to identify post-translational modifications.1
Advances on the EDT horizon include negative ion ETD, or reverse ETD, in which positively charged ETD reagent ions react with negatively charged analytes to induce fragmentation. That technique could be used for analyzing negative product ions such as carbohydrates and RNA rather than peptides, which are positively charged.
Outside the box
Wyatt Technology Corporation (Santa Barbara, Calif.) has applied laser light scattering principles to protein mobility through its Mobius instrument. That allows the investigator to measure the mobility of proteins smaller than the typical resolution limit of electrophoresis.
The Mobius can handle proteins as small as 2 nm, with an analysis time of 60 seconds or less. Additional options permit the measurement of hydrodynamic radius, through the use of backward scattered light.
Light scattering instruments not only measure protein molecular weight and hydrodynamic radius but also characterize the nature of protein conjugates or aggregates in order to determine subunit conformation and measure the stoichiometry of protein complexes.
The detector is not configured to separate proteins, so it requires a purified sample. Impure samples or samples with mixtures of proteins will return data that is an average of all of the molecules in the sample.
“It’s a noninvasive technique. Mass spec will break it down so it will only be able to measure molecular weight,” says Michelle Chen, head of analytical services, Wyatt Technology. Proteins analyzed by light scattering are not damaged or altered and can be used later.
No treatment of protein identification would be complete without discussing the immunoassay. The immunoassay is a workhorse of protein identification, coming in a wide range of formats, and applied across a spectrum of applications from bedside clinical applications to high content screening in the drug discovery industry.
Nanoscale immunoassays are a recent innovation that basically miniaturize the already-tiny microarray. NanoInk Inc., headquartered in Chicago, makes a dip-pen nanolithography platform that can generate nanoscale features on any solid surface. NanoInk has demonstrated the technology by developing a nano-scale ELISA array to detect recombinant prostate specific antigen. According to NanoInk, the miniaturized assay is capable of detecting prostate specific antigen (PSA) at femtogram per milliliter concentrations.
Nanoscale immunoassays “can be performed in a sandwich capture assay format as well as reverse phase array formats,” says Bruce Dudzik, senior director of Nano BioDiscovery Division at NanoInk. Comparing the dip pen nanolithography method to ink jet spotting, Dudzik said that the ink jet method subjects proteins to very high shear forces and may potentially disrupt the bioactivity of the protein. In contrast, the DPN deposition method produces highly uniform protein features.
A nanoscale assay requires significantly less sample than an ELISA, a bead-based assay, or a conventional microarray. The assay requires as little as 2 µL of sample, which makes it possible to carry out assays in situations where very little sample is available, such as needle aspirates from cancer patients or cerebral spinal fluid samples. The nanoscale assay may also be multiplexed in a single sample, further multiplying the value of the 2 µL sample.
“In many instances, the miniaturization process can also enable an increase in the sensitivity of the assay itself,” says Dudzik. That’s because concentration of the target molecules in a very small area in the nanoscale assay helps increase the kinetics of the reactions and brightens the detection signal registered from the assay.
Dudzik cautions that good laboratory technique must be used in performing the assay, because of the very small volume of sample. “Also, the assay does not fix a bad reagent,” he says. “For instance a poor affinity capture antibody will not be improved in our assay if it does not bind to its partner well to begin with.”
NanoInk makes a preformatted 10-plex human inflammation biomarker kit, and also provides custom biomarker assays. Past clients have requested assays for serum samples, whole blood samples, tumor lysates, tear samples, cerebrospinal fluid samples, and dried blood spots.
1. Lee AY, et al. “Quantitative analysis of histone deacetylase-1 selective histone modifications by differential mass spectrometry.” J Proteome Res. 2008:7(12);5177-86.
About the Author
Catherine Shaffer is a freelance science writer specializing in biotechnology and related disciplines with a background in laboratory research in the pharmaceutical industry.