Waters Develops New Label-Free, Mass Spec- Based Method for Absolute Quantification

Waters has developed a new label-free, mass spectrometer-based method for absolute quantification of proteins.

The new method, published in the Oct. 11 issue of Molecular & Cellular Proteomics, relies on an "unexpected" relationship between protein concentration and the average MS signal response for the three most intense tryptic peptides, according to Jeff Silva, the first author of the study who is a senior research and development scientist in Waters' Core Technologies/Proteomics department.

There is one important caveat to the method, Silva noted: So far, the method can only be performed using the Waters Q-TOF Premier mass spec, because the instrument has a special data-collection strategy that toggles back and forth between acquisition of low-energy precursor ion information and elevated energy fragmentation data.

Normally, the relative quantitation of a peptide as compared to another peptide can not be determined by comparing MS intensities because peptides ionize at different efficiencies, Silva explained. The new absolute quantification method is based upon a relationship that was discovered between absolute concentrations of proteins and the [MS signal] "response factor" of the three highest ionizing peptides of a given protein.

"It turns out that at equimolar concentration, the average intensity measurement of those top ionizing peptides are the same," said Silva. "So there's a counts-per-mole equivalent for any given protein."

Silva's research team tested the method of absolute quantification by first working with five proteins of known concentrations that varied in size from 15 to 98 kilodaltons, then carrying out the same calculations with a subset of human serum proteins of unknown concentrations.

The researchers calculated the average signal response from the top three tryptic peptides for each of the proteins, which varied in concentration from six picomoles to 15 pmoles. They showed that each of those proteins had the same response factor, such that the signal response is a fixed number of counts per picomole of protein.

Once they had the response factor for their Waters Q-TOF Premier instrument, the researchers used that factor to calculate the absolute quantity of 11 proteins in human serum. The concentrations of the proteins as determined by the new method correlated with concentrations of the proteins that had been previously published in other studies.

"It seems simple," said Minerva Hughes, a recent PhD graduate in Craig Townsend's laboratory at Johns Hopkins University who has been using the Waters Protein Expression system to do relative protein quantitation. "They've noticed a trend where if you look at the most abundant ions, you can generate a universal factor that you can multiply everything with. You find your scaling factor, and you can go back and actually figure out micromolar amounts."

The reason that only Waters Q-TOF Premier instruments work with this method is that so far, only those instruments collect data in an alternating fashion, such that there are three data points — mass, retention time, and signal intensity — per time interval, Silva said. Other instruments acquire data through only a fraction of the peak list, until they have gathered enough information to identify that peptide.

"The alternate scanning mode of acquisition allow you to get a true sense of what's there in terms of peak area to a given protein," said Silva. "Because we were able to see the true 3D peptide mass fingerprint, without partial peak sampling, the correlations fell right out of the data."

Silva and Hughes noted that absolute quantitation is important for determining the stoichiometry of molecules that make up a complex.

Paul Skipp, the manager of the Center of Proteomics Research at the University of Sussex in the U.K., added that absolute quantitation is important for understanding signaling pathways.

"In a signaling pathway, you tend to have a small signal that starts that causes a protein to bring other proteins to it — that may require 10 molecules, and those 10 molecules may signal to another protein," he said. "If you know a cell in its normal state has so many molecules of Y and so many molecules of Z, you start to understand how that cell's working. Also, if one of those molecules starts to produce a larger number of molecules, that could be an indicator of an onset of disease."

Skipp currently uses a technique called AQUA to do absolute quantitation. The method relies on using a ¹³C-labeled molecule as an internal standard. Skipp had not heard of Waters' new method of absolute quantitation, which does not require protein labeling, but said that he was pleased with the way Waters' Protein Expression system had worked for relative quantitation.

"I think the way that [mass spec] technology is going to move in the future is to move away from labeling," said Skipp. "The beauty of the Protein Expression is that you don't have to label, and you can compare more than four conditions, and you can compare different experiments to each other."

Silva said Waters is currently integrating the new absolute quantification relationships into their software so that it will be easy to calculate the average intensities of the top three ionizing peptides. He noted that the reason for using the top three ionizing peptides, as opposed to another number, was a function of the size of the protein. For small proteins, the top two might work better, and for large proteins the top four might work better, he said.

"You could make it a function of molecular weight, but three worked in this demonstration," he said.

— Tien Shun Lee ([email protected])