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UCSF Team Uses SRM-MS to Study Apoptosis via Global Kinetic Analysis of Proteolysis

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By Adam Bonislawski

Scientists at the University of California, San Francisco, have devised a selected-reaction monitoring mass spec workflow to measure the kinetics of caspase-driven proteolysis.

The technique has provided new insights into processes like apoptosis and cell division and could also be used to study the kinetics of other proteases and protein post-translational modifications like phosphorylation and acetylation, research team member James Wells, a professor in pharmaceutical sciences at UCSF, told ProteoMonitor.

As detailed in a study published online last month in the Proceedings of the National Academy of Sciences, the researchers developed SRM-MS assays for 676 peptides derived from cleavage of proteins by the human apoptotic caspases-3, -7, -8, and -9. Using an AB Sciex QTRAP 5500 instrument, they quantified these peptides in cell lysate at various time points, enabling them to simultaneously determine the intrinsic catalytic efficiencies of hundreds of caspase-substrate interactions.

According to Wells, who co-authored the study, several interesting findings emerged from these measurements. While researchers have published thousands of papers on apoptosis, the comparative kinetics of the targets involved has gone relatively unexamined, he said.

"What our data does is rank [the targets] in terms of their rates," Wells said. "What we found was that, amazingly, the rate at which these proteins are being cleaved varies by over 500-fold."

"Much to our surprise we found that among the proteins that are cleaved the fastest center around the microRNA processing as well as the endocytic pathway," Wells added. "And a lot of those targets overlapped among all the caspases – so they were hit hard, broadly, and by multiple caspases."

"We're still trying to figure out why that is, but it's something we could only get by understanding the kinetics of these processes," he said.

The researchers also found that inducing apoptosis with different agents – either staurosporine or TNF-related apoptosis-inducing ligand in the case of the PNAS paper – led to differences in cleavage rates for various proteins, Wells said.

Key to the study, he noted, was use of an N-termini labeling technique Wells and several colleagues detailed in a 2008 paper in Cell. Based upon enzyme engineering work Wells began while a staff scientist at Genentech, the technique tags α-amines in protein N-termini formed by proteolysis with biotinylated esters, allowing researchers to pull them out of a complex sample for mass spec analysis. Using this positive enrichment approach, Wells' team has been able to identify N-terminal peptides across six orders of magnitude in complex samples like cell lysate and serum.

In the PNAS work, the researchers pulled out and identified 1,341 caspase-cleaved peptides using LC-MS/MS on an Applied Biosystems QSTAR Elite mass spec. They then built SRM-MS assays for 676 of these peptides.

"A lot of work goes into validating these [SRM] transitions," Wells said. "It's pretty tedious. So, perhaps if we had chosen other transitions to focus on, maybe we could [have built assays for more peptides], but these [676] just came right out of the data analysis in a reliable and accurate way, so we felt like, 'OK, let's just focus on these and see what we get.'"

Wells estimated that his lab's N-termini tagging technique pulls out roughly two-thirds of all possible caspase-derived cleavage products, meaning that the method – even before taking into account peptides for which good SRM assays don't exist – doesn't provide a totally global profile of caspase kinetics.

"We know there are things we will miss," he said. "We'll miss things that have N-termini that are protected in the folded protein and can't be labeled with our enzyme-tagging method. We'll miss things that generate too short a tryptic peptide because they'll be removed during the gel filtration part of our sample work-up."

However, he said, he believes that the study is the first "to describe the kinetics en masse for proteolysis or any other post-translational modification in a complex milieu."

Since completing the PNAS work, the researchers have applied the technique to study caspase activity in a number of different cell types and in response to a number of different cancer drugs, and are preparing to submit another paper presenting the results of these efforts, Wells said.

"We've looked at a variety of different cell lines and a variety of different drugs to see how these change the spectrum of the kinetics," he said. "What we see is that in different cell lines we see different cleavage products coming up with different inducers at different rates."

"We want to understand what [proteins] are being [cleaved] most rapidly and ask if that correlates across different cell lines," Wells said. "What are things that are commonly seen things and rapidly cut things? Those may be important nodes."

Such nodes could prove key not only in apoptosis, but in cell differentiation as well, he added. "Caspases are activated during differentiation and then shut off. So it would stand to reason that the fastest nodes might be important in cell differentiation because with just a short burst of caspase activity, the caspases would go after their most appetizing substrates."

The researchers are currently using the N-termini tagging technique to investigate proteases outside the caspase family, Wells said, determining the substrates for a number of different enzymes.

SRM-MS could be useful for studying the kinetics of those interactions, too, he added, as well as for measuring the kinetics of other post-translational modifications like phosphorylation and acetylation.


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