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Max Planck Team Devises Streamlined, Single-chamber Method for Mass Spec Sample Prep

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Max Planck researchers have devised a streamlined mass spec workflow that allows researchers to perform sample prep from cell lysis through to elution of purified peptides in a single, enclosed volume.

Detailed in a paper published this week in Nature Methods, the method offers significant time savings as well as apparent improvements in reproducibility, Nils Kulak, a graduate student in the lab of Max Planck researcher Matthias Mann and author on the study, told ProteoMonitor.

Applying the method to analysis of yeast lysate on a Thermo Fisher Scientific Q Exactive instrument, the researchers identified 4,577 proteins, the most extensive proteomic profile of yeast reported to date, they said.

They used the workflow for a copy number analysis of HeLa cells, as well, obtaining copy numbers for 9,667 human proteins.

Sample prep for mass spec proteomics is typically a multistep process, in which proteins are extracted and solubilized from their source material and then denatured, reduced, and alkylated before undergoing digestion into peptides. Following digestion, the resulting peptide mixtures must then be cleaned up to remove various reagents that would hinder their analysis by mass spec.

Aiming to simplify this process, the Max Planck team either eliminated or substituted several reagents typically used in proteomic sample prep, allowing them to greatly compact the workflow. For instance, the researchers avoided using the detergent SDS, which must be removed from samples before digestion and LC-MS/MS. They also replaced the conventionally used reducing agent dithiothreitol and the alkylating agent odoacetamide with TCEP and choroacetamid, respectively. While the former pair of reagents react with one another and so must be added in separate steps, the latter pair can be combined, meaning that lysis, reduction, and alkylation can all be performed in a single step.

Additionally, reduction of these reactions to a single step meant that the researchers could combine them in a single chamber – in this case Thermo Scientific's StageTips, pipette tips with a built-in C18 disc.

In all, Kulak said, the researchers managed to reduce what had been a three to four hour process – not including the time required for enzymatic digestion – down to around half an hour.

And beyond the time savings, by reducing the number of manual steps involved and combining the reactions in a single chamber, the method also holds out the promise of less sample loss or contamination and improved reproducibility, Kulak added. He said that it was difficult to say precisely how much of an improvement in reproducibility the method offered, but noted that analyses the researchers had done using the new technique suggested reproducibility gains.

For instance, he said, analyses by the Max Planck team of biological replicates using the method had uncovered more statistically relevant differences in protein expression, suggesting "that the reproducibility within replicates has gotten better."

Kulak noted as well that in samples prepared as much as a year apart, the researchers saw correlations with R-squared values as high as 0.91, "which shows that it is really quite reproducible." He added that they had also achieved sensitivity improvements compared to conventional sample prep procedures.

Another apparent byproduct of the simplified workflow is improved retention of certain classes of proteins. In conventional procedures, cell lysis is typically followed by a clarification step, in which researchers spin down the insoluble fraction of the sample. The Max Planck researchers skipped this step and, Kulak said, observed an increase in the number of transmembrane and nucleic acid-binding proteins detected.

"What we saw is that this [clarification] step can actually remove certain transmembrane and nucleic acid-binding proteins," he said, adding that he, "personally, would not recommend clarifying anymore."

Kulak noted that while the Nature Methods study focused on analysis of cell lines, it could also be used for work in plasma – although, he said, researchers would still have to contend with the dynamic range challenges plasma presents.

The study, Kulak said, was perhaps indicative of a growing interest in simplifying mass spec sample prep. He cited a recent paper by the lab of University of Wisconsin-Madison researcher Josh Coon in which the authors similarly skipped lysate clarification, noting that "it seems recently people have started to think about skipping [sample prep] steps."

While the techniques and reagents employed by the Max Planck group have long existed, conventional sample prep procedures "were so established that most people never really thought about improving them," Kulak said. He suggested that this was in large part due to the fact that, given the extensive pre-fractionation traditionally required for deep proteome analysis, sample prep took what seemed to be a comparatively insignificant amount of time.

"People never really thought about reducing these [sample prep] steps to be faster because, especially with the very complicated pre-fractionation techniques, [mass spec-based proteomics] was so laborious anyway," he said.

However, with the appearance in recent years of mass spectrometers powerful enough to go deep into the proteome with little or no pre-fractionation, "it makes it extremely attractive to also make the sample prep faster," Kulak said.

He noted that the researchers have applied for a patent on the method with the ultimate aim of making it commercially available.

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