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Max Planck Team Develops Sample-Prep Method that Promises to Aid Study of Complex Proteomes

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This story originally ran on April 21.

Researchers at the Max Planck Institute for Biochemistry have devised a sample-preparation method combining the advantages of in-gel and in-solution digestion methods for mass spectrometry-based proteomics that they said could become a "crucial" technology for research into complex proteomes.

Using their approach, described in a study published April 19 in the online edition of Nature Methods, the researchers, which included Matthias Mann, identified 40,582 unique peptides corresponding to 7,093 proteins from HeLa cells — "to our knowledge … the largest reported proteome in any single experiment," they said.

As mass-spec technology continues to improve, more focus is now being directed at the front end of the proteomics workflow and, in particular, sample preparation is seen as significant a bottleneck in proteomics as instrument limitations.

In an interview with ProteoMonitor this week, the study's lead author and a member of Mann's laboratory at Max Planck, Jacek Wiśniewski, said that part of the issue is that different proteins have different properties that can affect their digestion. For example, he said, membrane proteins are highly hydrophobic and are not easily digested with in-solution methods.

Wiśniewski said the technology developed by him and his colleagues, called filter-aided sample preparation, or FASP, offers three main advantages: The first and most important is that it solubilizes everything from the biological material, using high concentrations of sodium dodecyl sulfate.

"You can solubilize all detergents … which was impossible until now using the in-solution approach," he said.

The second advantage is its speed. Depending on enzyme conditions, all samples can be prepared in two hours, plus the digestion time, with FASP. Many samples, he added, can be prepared in parallel even without automation.

And lastly, because of the purity of the peptide mixture that can be achieved with FASP, a liquid chromatography column can be reused for weeks.

"This is important in respect to the mass spectrometer because … when the peptide mixture is not clean and contains other components, the columns are [quickly clogged], and you have to change the columns frequently," Wiśniewski said.

The purity also leads to better data. "When you have other components that are not of a peptidic nature … in the mass spectrometer, you have signals, but the signals do not lead to identification of peptides," he said.

While the technology has commercial possibilities as a sample prep kit, he said that because the method can be applied to all types of proteomics research, all researchers can follow the protocol and implement it for their work.

'Proteomic Reactor'

FASP essentially combines elements from both in-gel and in-solution digestion strategies, while using a standard filtration device acting as a "proteomic reactor" for detergent removal, buffer exchange, chemical modification, and protein digestion, Wiśniewski and his colleagues wrote.

In-gel and in-solution are the main digestion approaches to proteomics research, but both have major drawbacks, the researchers said. In-gel digestion is robust and thus can safeguard against impurities that may interfere with digestion, "but the gel may prevent peptide recovery and cannot easily be automated," they said.

Meanwhile, in-solution digestion can be more readily automated and minimizes sample handling, but with such an approach, a proteome may be incompletely solubilized and interfering substances may impede digestion.

For total solubilization of cells and tissues, sodium dodecyl sulfate has been the reagent of choice, but as with any detergent, even in small amounts, SDS can "preclude enzymatic digestion and dominate mass spectra," Wiśniewski and his fellow researchers said. "Therefore, depletion of SDS is a prerequisite for efficient mass-spectrometric analysis in proteomics."

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In-solution removal of SDS has been considered impossible, so researchers have turned to other approaches for analyzing membrane proteomes. FASP was developed after the researchers discovered that membrane proteins "can be fully depleted from detergents by gel filtration in 8 M urea such that they can then be analyzed as efficiently as soluble proteins," they said.

They used this observation as a starting point to develop a method that would combine strong detergents for universal solubilization with a strategy to "clean up" the proteome before digestion. In such a method, purified proteins could be obtained after digestion while the drawbacks of in-gel digestion could be avoided.

They reasoned that a filtration device could be used as the clean-up mechanism.

After testing filters with relative molecular mass cut-offs of 3,000 and 10,000, they settled on a Millipore-Microcon YM-10 filter as their standard because it "efficiently retained small proteins" in the 5 kiloDalton to 10 kiloDalton range "and efficiently released peptides up to 5,000 Daltons."

In the Nature Methods article, the authors wrote that there are four critical steps to the FASP method, beginning with depletion of detrimental low molecular-weight component in urea-containing buffer. Other important steps include carboamidomethylation of thiols; the digestion of proteins; and the elution of peptides.

"Notably, during peptide elution, the filter retains high molecular-weight substances that would otherwise interfere with subsequence peptide separation," they wrote.

They first tested FASP for its efficiency and range of applicability. Various amounts of bovine serum albumin protein standard and total HeLa cell lysates were processed and analyzed. Based on UV-light absorption and LC-MS-MS analysis of BSA peptides, Wiśniewski and his colleagues determined that their approach "resulted in very high yield" — at least three orders of magnitude of protein abundance.

Then, they tested FASP on samples including mouse liver and brain tissues, as well as cultured cells. In single-run analyses with four-hour gradients, they identified 1,800 to 2,200 proteins with 99 percent confidence and at least two identified peptides per proteins using the MaxQuant algorithm.

Adding proteins that were identified with one peptide increased the number of proteins identified to between 2,200 and 2,700. In comparison, in a characterization of the liver proteome done by members of the Nature Methods team using extensive cytosolic and membrane fractionation with analysis of 20 in-gel slices, 2,210 total proteins were identified.

They also report that 75 percent to 80 percent of the fragmentation events in the FASP dataset resulted in the identification of the peptide in the database. Such high rates of identification have been achieved only in stable isotope labeling with amino acids in culture pairs, "suggesting that the high purity of eluted FASP peptides minimized fragmentation events associated with chemical noise, which cannot lead to peptide identifications," they said.

Furthermore, Gene Ontology analysis indicated 42 percent of HeLA total cell lysate proteins and 52 percent of brain tissue proteins matched to the membrane category, indicating the absence of bias against hydrophobic proteins compared to soluble proteins, Wiśniewski and his co-researchers wrote.

Largest Proteome

In previous work, Wiśniewski and colleagues reported identifying 22,905 peptides from 3,979 proteins from HeLa cells in a workflow that combined peptide isoelectric focusing in Agilent Technologies' Offgel Fractionator with 12 peptide fractions and in-solution digestion. Using FASP, they were able to increase identification to 40,582 peptides corresponding to 7,093 proteins from HeLa cells, which they said is the largest proteome they know of ever identified in a single experiment.

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In continuing work, Wiśniewski said the coverage has further increased to about 50,000 peptides corresponding to close to 10,000 proteins.

"People consider that HeLa cells have probably 12,000 gene products, proteins, so we're very close to the complete proteome using the FASP approach," he said.

Using Gene Ontology analysis, they said, they determined the FASP-prepared proteome was not biased for proteins from any compartments or protein classes, but instead "the FASP preparation method is universal in that it does not lead to preferential extraction of proteins from specific cellular compartments or with specific functions."

FASP also offered advantages over the researchers' previous HeLa experiments in low-abundance protein identification. The percentage of proteins annotated by Gene Ontology for transcription, signal transduction, and receptor activity increased by 20 to 30 percent, they researchers reported, and they identified more than 90 percent of the proteins involved in the oxidative phosphorylation pathway assembly of the ribosome, RNA polymerase, and the polymerase II transcriptional machinery.

"Considering that some of these proteins were cell type-specific and cell stage-specific, and therefore were not expressed in all conditions, our data had very high coverage," the researchers wrote.

Finally, they said, the ability to identify more than 2,000 proteins in single runs using only 1 to 2 micrograms of material "opens up interesting applications for proteomics..." In organelle analysis, for example, FASP offers at least an order of magnitude improvement in sensitivity and protein-identification number than other proteome techniques such as 2D-gel analysis.

"For in-depth analysis of complex, mammalian proteomes, FASP could be a crucial enabling sample preparation technology," they said.

Gang Chen, a professor of analytical chemistry at the School of Pharmacy at Fudan University in Shanghai, China, called the method "useful" and said that he is interested in using elements of FASP in his own work designing a microfluidic bioreactor for proteolysis. "Maybe we can integrate the trypsin-immobilized ultrafilter in the channel to fabricate [a] filtration unit for on-chip sample preparation," he said.

But, he added, the Nature Methods paper is a technical note that needs further validation. "I would like to see someone use this method," he said.

Wiśniewski said, though, that the method is already being used by others at Max Planck and he is using FASP as the standard sample-prep method in his proteomics research.

"In fact the method is ready to use, there is no need to develop it [further]," he said, though he and his co-researchers are developing it for use in combination with other methods. For example, they are developing it for affinity chromatography "so we can … enrich for phosphoproteins, or glycoproteins … without using any special resins where the ligands are immobilized," he said.

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