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TUM Study Suggests Addition of DMSO to LC-MS Runs Could Significantly Up Peptide IDs

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According to a study led by researchers at Technical University Munich, the addition of an inexpensive solvent to electrospray ionization mass spectrometry runs could offer a significant improvement in peptide identification by LC-MS/MS.

In a paper published this week in Nature Methods, the researchers demonstrated that adding 5 percent DMSO to the solvents used in the upfront LC separation increased the mass spec signal intensity of the measured peptides by a median factor of three and improved the sensitivity of detection by as much as 10-fold.

Given the low price of DMSO and the relative ease of adding it to an LC run, the technique could prove a broadly applicable enhancement to mass spec-based proteomics, TUM researcher Bernhard Kuster, senior author on the study, told ProteoMonitor.

"It is so exceedingly simple to do that anyone operating an LC can just add DMSO to their solvent," he said. "It costs a penny to do."

As Kuster noted, the TUM team's efforts build on a study published last year in the Journal of The American Society for Mass Spectrometry by University of California, San Diego researcher Elizabeth Komives that first identified DMSO as enhancing peptide identification via LC-MS/MS.

In that research, Komives and her colleagues observed that the addition of DMSO led to what they termed "charge-state coalescence," in which lower and higher charged peptide precursor ions were diminished with a single primary charge state dominating instead. This phenomenon leads to a simpler precursor ion spectra, which in turns leads to more clear-cut mass spec data and peptide IDs.

In an email this week to ProteoMonitor, Komives said that the TUM researchers moved her lab's findings forward by observing that the addition of DMSO improved in particular the analysis of "low abundance peptides and complex mixtures." She noted that her team had not been able to investigate this angle due to their "inferior instrumentation."

Indeed, Kuster and his colleagues found that addition of DMSO improved detection of low-abundance peptides by a median of six-fold versus roughly three-fold for high-abundance peptides. The TUM researchers, however, found that only a small proportion – roughly 10 to 20 percent – of the improvement was attributable to charge-state coalescence, observing instead that most of the enhancement was due to more efficient production of ions during the electrospray process.

In the Nature Methods paper, the authors theorized that the addition of DMSO leads to faster and more complete "sequestration of single peptide molecules into charged nanoscale droplets" and that this sequestration of single peptides increases their "likelihood of ionization."

"If you just have one [peptide] molecule in one droplet that is highly charged, there is a good chance it will ionize," Kuster said. If, on the other hand, you have larger droplets containing a large number of molecules, there will be competition for ionization among the many molecules in that droplet, he noted.

In that case, Kuster said, "there will be a bias towards the [peptides] that are most evenly charged, which will be the ones that are basic to start with [given] the acidic conditions under which one does reverse phase chromatography."

The TUM researchers tested the technique on a range of instruments, including a Thermo Fisher Scientific LTQ-Orbitrap XL, an Orbitrap Velos, an Orbitrap Elite, and a Waters Synapt G2. They also had colleagues at the Swiss Federal Institute of Technology, Zurich try it on an AB Sciex TripleTOF 5600 and researchers at Waters test it on several of that company's Q-TOF instruments, Kuster said, noting that it worked on all the machines used.

"We really do think that it is a general effect," he said.

According to Kuster, the TUM team has not observed any effects from adding DMSO that would significantly hamper the approach's utility, though he noted that because the solvent is a fairly strong eluent, researchers may need to adjust their chromatography gradients to account for this effect.

Kuster said that, at least in his lab's work, this potential inconvenience was countered by the fact that the addition of DMSO cleaned "the LC columns quite nicely, so the carryover from sample to sample is greatly diminished."

He noted that while the technique appears to be broadly applicable to peptides, some tweaks might be necessary for working with strongly hydrophilic molecules, such as some glycopeptides. Very hydrophilic peptides could "on the one hand, benefit from the improved ionization" provided by the addition of the DMSO, Kuster said. However, addition of the solvent could "on the other hand, wash them off the reverse phase [LC] column, which would not be so good."

This issue might be addressed by using a different type of column material – such as graphitized carbon – that would better retain such peptides, Kuster said. Researchers could also add the DMSO in between the LC and ionization steps, although he noted that this second approach could lead to dead volume that would lessen the resolution of the LC.

The TUM researchers have not patented this use of DMSO, Kuster said. In the first place, he noted, the fact that the initial discovery was made by Komives' lab would likely make any patent difficult to defend. Beyond that, he said, "the benefit would just be so nice to have that it would be a disservice to the community to stop people from using it."

In a similar vein, Kuster noted that given the simplicity of the technique and the low cost of DMSO, it was unlikely to offer much of a sales opportunity for LC or mass spec vendors.

"There's not really a business to be made, I don't think, in selling mass spec grade DMSO," he said.

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