Laura Lane has worked as a health and science journalist since 1997. She received her master's degree in biology from Stanford University. Since then, she has written for the Dallas Morning News, the Contra Costa Times, Shape magazine, WebMD, Yoga Journal, Diagnostic Imaging, the International Medical News Group, The Scientist, Bio IT World and Biocompare.
Recovering from cocaine addiction requires monumental effort. Without a drug that specifically prevents craving for the white powder, addicts need plenty of stamina, strength and support to endure withdrawal symptoms and reject the insistent cravings for more.
Fortunately, researchers have made significant progress in understanding the molecular mechanisms underlying drug addiction, which, in turn, open the way towards new therapeutic strategies. And some are achieving that by focusing on post-translational modifications (PTMs).
For example, a team of researchers examined and quantitated the phosphorylation states at individual sites in the transcription factor ΔFosB. Turns out, it’s phosphorylated, and in turn stabilized, by the protein kinase CaMKIIα in response to cocaine. Phosphorylated ΔFosB goes on to upregulate transcription of the CaMKIIα gene, increasing ΔFosB accumulation and leading to further CaMKIIα induction [1]. Disrupting this event could help treat addiction.
Such discoveries hinge on the ability to purify samples, enriching for target proteins and peptides and isolating them from others. In theory, sample purification for studying PTMs isn’t really all that different from any other project. However, it does require understanding the chemical nature of PTMs, and the proteomic environment in which they exist.
World of diversity
Staff in Yale’s W.M. Keck Foundation Biotechnology Resource Laboratory and its associated Yale/NIDA Neuroproteomics Center handle most of Yale's requests for analyses focused on PTMs, including those involved with the cocaine study. TuKiet Lam, Director of the Posttranslational Modifications Core in the NIDA Center offers the following pointers for some of the more common PTMs:
Phosphorylation: Use titanium dioxide (TiO2) metal-oxide affinity chromatography, reagents for which you can find from several vendors. These resins selectively bind to phosphopeptides. However, titanium dioxide tends to bind more reliably to peptides with more than one phosphorylation, while immobilized metal-ion affinity chromatography (IMAC) may be more reliable for monophosphates. In global phosphorylation studies, researchers often utilize a combination of chemistries including TiO2, IMAC, and others, such as ZrO2.
Glycosylation: Lam recommends separating glycosylated proteins prior to trypsin digestion. Use affinity-based chromatography with lectins such as concanavalin A or wheat-germ agglutinin, which bind to glycans. Then, treat with peptide-N-glycosidase F (PNGase F, available from New England Biolabs and other vendors), which cleaves N-linked carbohydrates from residues such as asparagine, leaving aspartic acid in its place – a chemical modification that can be detected with mass spectrometry.
Ubiquitination: Use an antibody to separate out ubiquitinated proteins directly, or digest with trypsin and purify the now de-ubiquitinated peptides with an antibody that recognizes what’s left of the ubiquitin linkage. Trypsin digestion of ubiquitinated protein destroys the ubiquitin itself, leaving a di-glycine motif on the modified lysine. Modified peptides can be purified with an antibody recognizing that motif and detected using mass spectrometry. (Cell Signaling Technologies’ PTMScan® Ubiquitin Remnant Motif (K-ε-GG) Kit is one commercial form of this assay.)
Palmitoylation: Again, Lam suggests separating out palmitoylated proteins first. Block free sulfhydryl (-SH) groups with N-ethylmaleimide, then cleave the fatty acid from the protein with hydroxylamine and introduce biotin (a process called acyl-biotinyl exchange [2]). Use an avidin column, which recognizes biotin, to enrich for the formerly palmitoylated peptides. Finally, digest the purified proteins with trypsin and analyze by mass spectrometry.
The needle in the haystack
Although Lam has strategies for many other PTMs up his sleeve, you also need to consider the specific tools for accurately separating, enriching and quantifying post-translational modifications. Most often, this involves “find[ing] that needle in the haystack, even if the haystack is still there,” says Angus Nairn, co-director of the Yale/NIDA Neuroproteomics Center.
As with many other protein studies, column chromatography is your best bet for finding that metaphorical needle, and the past several years have seen major progress on that front, says Ken Cook, bioseparations manager at Thermo Fisher Scientific.
“We’re at the stage now where companies can do small molecules really well,” Cook says. “Now we’re moving into exciting areas in the protein world with larger molecules.”
One of the biggest advances is the dramatic reduction in particle size of resins that pack the columns. Once commonly 10 microns, resin particles are now below three microns in size. Smaller resins provide more surface area for protein binding and pack into the column tighter, leaving less space in between particles.
“As you go smaller and smaller in particle size you reduce the diffusion distance in the porous particle so you get sharper peaks, which means better performance,” says Michael McGinley, bioseparations product manager at Phenomenex.
Smaller particle size, however, causes pressures in the column to increase. In response, companies have designed columns to handle higher pressures. You can now find columns, such as Thermo Fisher’s new MP35N columns, that can withstand more than 15,000 psi.
DIY chemistry
You can bring more precision into your protein purifications by using affinity ligands for the specific PTM that you’re studying. But with so many possible PTMs and attachment sites, even within one protein, you might need to produce your own beads with the phospho-specific antibody that exactly suits your investigation.
EMD Millipore’s PureProteome™ NHS FlexiBind Magnetic beads offer an easy way to accomplish this task. The product provides magnetic beads with the chemistry required to attach antibodies or other amine-containing biomolecules.
“You don’t need to have [a] Ph.D. in chemistry to do the work,” says John Moskow, technology development manager at EMD Millipore. “You don’t have to wait for someone to offer your protein or antibody on their bead.”
And with magnetic beads, Moskow explains, you very efficiently pull the beads out of solution, providing a good option for working with smaller volumes and recovering low-abundance proteins.
Full of inhibitions
To get an accurate picture of which proteins carry which PTMs in the cell’s natural state, you’ll need to use a healthy dose of enzyme inhibitors during purification. Because phosphorylation is the most common PTM studied, Sigma Aldrich offers a wide selection of protease, phosphatase and kinase inhibitors, packaging them in different combinations in inhibitor cocktails.
“We try to hit as many deleterious enzymes as possible in the cell extract,” says Bob Gates, market segment manager at Sigma Aldrich.
Researchers can opt for broad-range inhibitors or inhibitors targeting more specific PTM sites. Adding or subtracting different inhibitors can provide different results on the patterns of proteins that are phosphorylated and at which sites. This offers clues as to how the target protein interacts with various phosphatases and kinases.
It’s an issue of “which proteases or phosphatases need to be [inhibited],” says Aaron Sin, also a market segment manager at Sigma Aldrich.
“It’s a discovery question,” he explains. “We don’t know which are important.”
Even with the best of tools and kits for purifying proteins, studying PTMs poses great challenges. Most notably, says Yale’s Nairn, there’s the problem of multiple modifications on individual proteins, an understanding of which will come only with monumental effort.
“Virtually every protein that’s phosphorylated is phosphorylated at multiple sites,” he says. “In many cases, we don’t know the combinatorial possibilities.”
Thanks to existing PTM-analysis tools, however, researchers have the means to figure it out.
References
[1] Robison, AJ, et al., “Behavioral and structural responses to chronic cocaine require a feedforward loop involving ΔFosB and calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell,” The Journal of Neuroscience, 33(10):4295-4307, 2013. [PubMed]
[2] Wan, J, et al., “Palmitoylated proteins: Purification and identification,” Nature Protocols, 2(7):1573-84, 2007, 2007. [PubMed]