news
Hunting the elusive d-amino acid
Relative intensity
Relative intensity
Sometime during the history The researchers first tested (a) 691 of life on this planet, l-amino the enzyme’s activity with repacids became the enantiomer resentative peptides, some of 331 of choice for building proteins. which had a single d-amino acid substitution. The rates of There doesn’t seem to be a 465 digestion for the l-amino acid particular reason this configupeptides ranged from a few ration was selected; scientists m/z minutes to an hour (influenced have built proteins from excluby certain l-amino acids or sively d-amino acids and found (b) 465 that they fold just like their posttranslational modifications natural counterparts, albeit as that are degraded more slowly a mirror image of the native than others), but the peptides structure. with a d-amino acid were essentially resistant to degradaAlthough proteins made tion by the aminopeptidase. entirely from d-amino acids do m/z not exist in nature, some proAs a proof of principle for a MS spectra showing that (a) before digestion with alanyl aminoteins and peptides do contain more complex mixture of peppeptidase, NWFa/NdWFa peaks (m/z 465) are barely detectable a small number of d-amino tides, they used a sample from above the noise, but (b) after digestion, the NdWFa peak is clearly the abdominal ganglion of the acids. For many peptides, this enantiomeric switch is thought visible. Tandem MS experiments confirmed the identity of both mollusk Aplysia kurodai, compeaks. to occur as a late-stage postmonly known as the sea hare. translational modification of The researchers compared a tivity, so DAACPs are easily overlooked. an l-amino acid in the peptide. sample that had been digested with the The true number of d-amino-acid“Some of these putative neuropeptides aminopeptidase with one that had not containing peptides (DAACPs) is not may be working by targeting a particuundergone enzymatic treatment. On known, because distinguishing peptides lar receptor, others may have very subtle comparing spectra from the two samthat contain only l-amino acids from effects, some may do nothing,” Sweeples, they found a peak in the treated those that contain d-amino acids is not dler explains. “If you don’t have potent sample consistent with NdWFa, a peptrivial; each peptide of interest must bioactivity, it’s hard to know if you tide containing a d-amino acid that is a known constituent of cells from this sysbe examined individually, which can made the wrong molecule.” tem; this confirmed that DAACPs could be extremely time-consuming. Now, The researchers wanted to find a way be isolated from a complex mixture. in work published in AC (2008, 80, to pick out the DAACPs before they Sweedler says that his group is 2874–2880), Jonathan Sweedler and went to the trouble of synthesizing the now hunting for previously unknown colleagues at the University of Illinois peptides. “Our goal here was to make DAACPs. Although the enzyme that Urbana–Champaign have devised a an approach that would be general— carries out the conversion from an l- to faster and simpler MS method to scan that would allow you to take a complex for potential DAACPs in a complex bio- sample of brain homogenate and say that a d-amino acid is not known, the position of the d-amino acid two to three logical sample. [a particular peptide] is more likely to In a peptidomics experiment, a sam have a d-amino acid in it,” says Sweedler. residues from the N-terminus is conTo do that, the scientists took adsistent across peptides from mollusks, ple (in this case, brain tissue) is fracvantage of the specificity of alanyl ami arthropods, and vertebrates; this sugtionated, and hundreds of peptides are nopept idase, an enzyme that digests gests that a common enzyme is makisolated and sequenced. A few selected ing this modification. “Most enzymatic peptides are then synthesized and tested proteins with a d-amino acid modification much more slowly than those made pathways that are this well represented for bioactivity. The catch is that any completely of l-amino acids. Sweedler may be found everywhere,” says Sweeof the native peptides could contain a d-amino acid—which wouldn’t be oband colleagues postulated that if they dler. “So it’s an interesting thought vious from typical MS data—and the added the aminopeptidase to a mixture to see if these are found in the mamresearchers could be unknowingly synof peptides, it would digest away the se- malian brain.” Now that searching for thesizing the wrong molecule. quences that contain only l-amino acids DAACPs has become simpler, it’s defiTo further complicate the experiment, and leave the peptides that contain a dnitely worth a look. a —Jennifer Griffiths amino acid. not every peptide will have biological ac3070
A n a l y t i c a l C h e m i s t r y / Ma y 1 , 2 0 0 8
