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Cornell Researchers Push the Limits for Identifying Posttranslational Modifications


Analyzing proteins for posttranslational modifications using mass spectrometry is a tricky business, usually requiring many rounds of analysis of peptide fragments. Wouldn’t it be nice to be able to stick an entire protein into the mass spec and get out all of the modifications?

Now, some researchers have come closer to this goal. In a proof-of-concept study, Fred McLafferty and colleagues from Cornell University have demonstrated that they can identify virtually all the posttranslational modifications in an intact protein of 29 kDa.

Although the technique may not see widespread application for several years, McLafferty’s research shows that it one day may be possible to determine the type and location of every posttranslational modification taking a few mass spectra.

In an article published in this week’s Proceedings of the National Academy of Sciences, he describes cleaving 250 of the 258 peptide bonds of the protein carbonic anhydrase, using electron-capture dissociation combined with electrospray Fourier transform ion cyclotron resonance mass spectrometry. Unlike most researchers, McLafferty ionized the intact protein, rather than first digesting the protein in a protease cleavage step. Previously, the largest protein analyzed in this way had been ubiquitin, which has 76 residues.

Electron-capture dissociation, the fragmentation method used in this approach, cleaves the protein backbone specifically and almost randomly, leaving other bonds — including non-covalent ones — intact, McLafferty said. He and his colleagues optimized the experimental conditions such that “we can get essentially complete sequence information and essentially complete information on where posttranslational modifications have taken place.”

“It’s a neat contribution,” said Donald Hunt, a protein mass spectrometrist at the University of Virginia. Hunt was especially intrigued by the fragmentation method. “[Electron-capture dissociation] is truly unique,” he said, because it only cleaves the protein backbone, leaving posttranslational modifications intact. “Most likely it has tremendous potential if applied to tryptic peptides.”

But despite its promise, McLafferty’s technique is far from becoming mainstream anytime soon. Although the fragmentation method is precise, Hunt cautioned that “nobody has yet got it to proceed with anywhere near the efficiency of the other fragmentation pathways.”

In addition, the approach has so far only been used with a 29 kDa protein, excluding larger ones. Another problem was the amount of protein McLafferty used in his experiments. According to an estimate, he used hundreds of picomoles of pure protein — a far cry from the small amounts found in complex biological samples. Furthermore, only a small fraction of intact proteins can undergo electrospray ionization, limiting the technique’s applicability.

“If you put it in perspective of where the field is at the moment, it’s not competitive,” Hunt cautioned, adding “but most things, when they start out, aren’t.”

McLafferty, on the other hand, pointed out limitations of the proteolysis approach relied on by most researchers. Not every protein fragment is usually recovered, he said, and unlike ions created by electron capture dissociation, the fragments often do not include an N- or C-terminus, making identifications of posttranslational modifications more difficult.

Hunt added that the proteolysis approach might also miss modifications that affect only a small percentage of protein molecules in a sample, whereas studying an intact protein would not.

McLafferty wants to improve his method further to run “many more proteins” and “very small samples on a routine basis.” Apart from determining posttranslational modifications, he said, there are other applications, such as studying protein folding in the gas phase.

— JK

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