Clustering is key to lighting up the dark proteome

Enlarge

Most mass spectrometry studies attempt to identify Peptide-Spectrum Matches (PSMs) and often ignore Spectrum-Spectrum Matches (SSMs), especially if PSMs for these SSMs are not established. However, SSMs also are useful when the corresponding peptide is not identified, because they allow a researcher to cross-reference spectra generated by different researchers and to query all spectra ever generated against a single repository. Spectral libraries are essentially databases of PSMs, while spectral archives are databases of both PSMs and SSMs. Although construction of PSMs (via tandem mass spectrometry database search) is a well-studied topic, construction of all SSMs represents a formidable clustering problem. The figure reveals similarities and highlights differences between construction (left) and use (right) of spectral libraries and spectral archives. With an archive, researchers first cluster, then search the clusters against a protein database to generate Peptide-Cluster Matches (PCMs). In turn, these PCMs get propagated to all spectra in the identified clusters to generate PSMs. With the library, researchers first search the spectra against a protein database to generate PSMs, group PSMs corresponding to the same peptide, and finally deposit the curated consensus PSM in the spectral library. Then, the spectral library can be used to identify spectra from new spectral datasets.

A new approach that organizes previously unused mass spectra from proteomics studies gives scientists the ability to use these spectra to gain more information about proteins in a wide range of organisms. Scientists from the University of California-San Diego and Pacific Northwest National Laboratory have created a vast spectral archive from more than a billion mass spectra acquired at PNNL between 2001 and 2009. They describe their approach in the July issue of Nature Methods.

In recent years, the volume of tandem mass spectrometry data generated from proteomics experiments has increased dramatically. Multiple, nearly identical mass spectra of the same peptides are routinely measured by various laboratories. Scientists compare the spectra with peptides residing in a database of known protein sequences. They then evaluate the resulting matches using various scoring methods to assign an identity to the peptide spectrum. Large sets of spectra can be organized into spectral libraries where other spectra can be brought for comparison, leading to increasing effectiveness in peptide assignments used for protein identifications.

But what about those spectra not identified; that is, those not associated with a known peptide? Typically, unidentified spectra are ignored or discarded, as they have limited value to the researchers because the protein is unidentified. As a result, a significant fraction of the proteins remain unidentified, constituting an effective "dark proteome" of unknown content.

Shedding light on the dark proteome is where the UCSD/PNNL team comes in. While spectral libraries discard unidentified spectra, spectral archives use all mass spectra—identified or unidentified-as clusters (see "Spectral Archives Complement Spectral Libraries"). The scientists not only showed the feasibility of constructing large archives and their basic utility for run-of-the-mill peptide identification, they developed new applications now possible because a diverse collection of datasets can be analyzed as a whole.

"We believe that spectral archives could change the nature of proteomics by motivating researchers who are analyzing seemingly unrelated data to share this data," said senior author Dr. Pavel Pevzner, UCSD. "Doing so improves the quality of the interpretations of both of their spectral datasets."

With archives, a researcher can identify clusters of spectra from different organisms. Besides indicating that such spectra are interesting—as they are likely to indicate proteins occurring over multiple species—this fact can be used to reduce the effective protein database size, leading to new, confident peptide and protein identifications. The team also showed that short peptides (shorter than 7 amino acids) could be confidently identified, which is much more difficult with typically used approaches.

The PNNL mass spectra data used by the team included samples taken from a diverse set of more than 100 organisms, including humans, the common house mouse, and the metal-reducing bacterium Shewanella oneidensis. The research team developed a clustering tool, MS-Cluster, that generated a spectral archive from the ~1.18 billion spectra from PNNL. This archive greatly exceeds the size of existing spectral repositories.

To evaluate whether spectral archives can increase peptide identifications, the researchers selected a subset of 14.5 million spectra from the microorganism S. oneidensis and constructed an archive with them. They did this by breaking the dataset into five sets of ~2.9 million spectra then incrementally adding each set of spectra to the archive. At each stage they compared the number of protein and unique peptide identifications made by searching the clusters in the archive with the number that could be obtained with conventional database search approaches.

The archive consistently yielded more unique peptide and protein identifications. With the archive, the scientists also were able to identify many more spectra through their cluster membership. At different stages, they identified 50-75% more spectra through cluster membership than via a regular database search.

This study also highlights the large number of spectra for which peptide and protein identifications are not achieved, opening the door for use of experimental and computational approaches to identify the significant numbers of peptides effectively ignored by proteomics studies to date.

More information: Frank AM, et al. 2011. "Spectral archives: extending spectral libraries to analyze both identified and unidentified spectra." Nature Methods 8(7):587-591. DOI:10.1038/nmeth.1609

Provided by Pacific Northwest National Laboratory (news : web)

Market lighting affects nutrients

Many people reach toward the back of the fresh-produce shelf to find the freshest salad greens with the latest expiration dates. But a study led by U.S. Department of Agriculture (USDA) scientists may prompt consumers to instead look for packages that receive the greatest exposure to light--usually those found closest to the front.

The study was led by postharvest plant physiologist Gene Lester while at the Agricultural Research Service (ARS) Crop Quality and Fruit Insects Research Unit in Weslaco, Texas. ARS is USDA's chief intramural scientific research agency.

Lester and colleagues Donald Makus and Mark Hodges found that spinach leaves exposed to continuous light during storage were, overall, more nutritionally dense than leaves exposed to continuous dark. Lester now works at the ARS Food Quality Laboratory in Beltsville, Md.

For the study, the researchers exposed spinach leaves to light similar to the 24-hour artificial fluorescent light received by spinach in packages located at the front of the display case. A second group was enclosed in two-layer-thick, brown-grocery-bag paper to represent the "dark treatment."

Both experimental groups were housed in market-type, light-transmissible polymer tubs with snap-tight lids and were kept in walk-in storage chambers at 4 degrees Celsius, the same temperature at which markets currently display packaged spinach. The light reaction of photosynthesis is not temperature-dependent and can occur at 4 degrees C in the right type of light.

The researchers found that the continuous light affected the leaves' photosynthetic system-resulting in a significant increase in levels of carotenoids and vitamins C, E, K, and B9, or folate.

While the simulated retail light conditions actually helped the stored leaves gain in content of several human-healthy vitamins, some wilting occurred after three days of storage in flat-leaf spinach, but not crinkled-leaf types.

Results from this work were published in the Journal of Agricultural and Food Chemistry.

Provided by United States Department of Agriculture

Lighting up a protein called Spy

 James Bond frequently has to undertake spectacular feats to protect Queen and country against utter destruction under insurmountable odds. But what happens when the homeland is a bacterial cell, and the danger comes from the insurmountable odds of making large amounts of a complex molecule? You call in a sleeper agent – a protein called Spy.

That’s the discovery made by biochemist James Bardwell and his team from the University of Michigan, Howard Hughes Medical Institute, McGill University and the National Research Council of Canada’s Biotechnology Research Institute. Their findings, which included a model of the protein obtained from the Canadian Light Source, were published in the journal Nature Structural and Molecular Biology.

Many proteins used in pharmaceuticals, such as insulin, can be manufactured by bacteria that have had the instructions for making the desired protein inserted into their genetic code. However, the process doesn’t work well for all proteins, leading to botched batches of poorly-folded molecular clumps that are unable to function properly. Bardwell and his colleagues wanted to see if the system could be improved by making the bacteria an offer that was hard to refuse – linking protein production to their survival.

“We gave the bacteria a pretty stark choice – fold proteins or die,” explained Dr. Bardwell. “We linked making a stable target protein in strains of E. coli to their resistance to penicillin, and then grew them in a medium containing the drug. The bacteria could survive only if they could also fold the target protein really well.”

The result: surviving bacteria produced up to 700 times more of the desired target protein than they would normally, along with large amounts of a little protein called Spy (short for spheroplast protein Y). It turns out Spy is a molecular chaperone – a type of molecule that facilitates the folding of proteins. The amount of Spy normally present in cells is vanishingly small, but in response to stress it is made in huge amounts . It is particularly induced in response to threats that would cause proteins inside the bacterium to unravel or clump uselessly, such as alcohol or tannins. While Spy had been previously identified inside bacteria, its function wasn’t understood until it was implicated in these ‘fold or die’ experiments.

“A lot of people were surprised that such well-studied bacteria as E. coli had a chaperone that had remained undiscovered,” said Dr. Bardwell. “But many assay tests that look for chaperones wouldn’t find Spy; then again, no one else has tried to genetically select for protein folding like we did.”

To better understand the workings of their enigmatic chaperone, the team needed to determine Spy’s molecular structure. Dr. Bardwell turned to his collaborator, Prof. Mirek Cygler at McGill University and the NRC Biotechnology Research Institute, who in turn sent crystals of Spy to the CLS.

The molecular model obtained from the CLS data revealed that Spy had even more surprises in store. Spy is one of the thinnest chaperone molecules ever found – a cradle-shaped molecule consisting of two parts, only nine ten-millionths of a millimetre thick (the same thickness, roughly, of nine hydrogen atoms) or about one fortieth the size of some better known chaperones. Spy’s cradle-shape may allow it to surround larger proteins, enabling them to fold properly while protecting them from harmful influences.

“It kind of acts like Teflon or a candy wrapper, covering the proteins and keeping them from clumping and sticking together,” Dr. Bardwell noted.

Now that Spy has been identified, Dr. Bardwell and his team plan to study how the protein works in detail, while also looking for similar chaperones using the same genetic selection routine. They also hope to force bacteria to produce other poorly folding, unstable proteins like HIV’s ‘tail protein,’ seen by many as the target for a potential AIDS vaccine.

“Spy is the way E. coli fights off stressful environments by protecting proteins from unfolding,” he explained. “By making Spy fold the proteins we want, there are lots of places we could go.”

More information: Quan et al. 2011. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. Nature Stuctural and Molecular Biology DOI:10.1038/nsmb.2016

Provided by Canadian Light Source