LONG BEACH, Calif. A study on relative protein quantitation by the Association of Biomolecular Research Facilities' Proteomics Research Group found that label-free mass spectrometry methods and multiplexed gel electrophoresis methods performed comparatively well in determining the relative quantitations of two unknown protein mixtures.
Non-multiplexed gel methods, such as Coomassie and silver stained gels, on the other hand, performed relatively poorly in the quantitation study.
"This was an eye opener," said Chris Turck, the chair of the PRG who presented results of the study here this week at the annual ABRF conference. "I was surprised about the label-free [mass spec methods] how well they did."
Turck cautioned, however, that results should take into account that some people are new to the area of quantitation, and the lack of experience may have hindered their success.
"[This study] doesn't mean that everybody doing Coomassie is getting high error rates [in quantitation]," said Turck, who is also the head of the clinical proteomics group at the Max Planck Institute of Psychiatry.
Members of the PRG decided to make their first-ever study on quantitation "not too difficult," said Turck. They sent out two mixtures containing eight proteins each to each of the participating laboratories. The two mixtures contained identical proteins. Four of the proteins had equal amounts, or 1:1 ratios, in both mixtures. The remaining four proteins had ratios of 1:3, 1:4, 1:5, and 1:76.
"This was an eye opener. I was surprised about the label-free [mass spec methods] how well they did."
Participants were given some information about the mixtures. They were told, for example, that four out of eight of the proteins were present at a 1:1 ratio, and that bovine serum albumin was among those four proteins. In addition, participants were told that there was a total of about 80 micrograms of protein in the mixtures, and that the dynamic range of the proteins was not greater than 1:100.
Ninety-two sets of protein mixtures were sent out to laboratories, and 52 laboratories returned results. Twenty-five of the laboratories were academic, 11 were governmental, six were vendors or manufacturers, and one was non-profit. Thirty-three of the participating laboratories were in North America, 14 were in Europe, and five were in Asia or Australia.
When asked what kind of method they used to perform relative quantitation on the samples, 36.7 percent of participants said they used electrophoresis-based methods, including 1D and 2D Coomassie-stain gels, fluorescence gels, and silver-stain gels; differential gel electrophoresis, or DIGE; and 2D differential radioactivity gels.
The remaining 63.3 percent of participating laboratories used mass spectrometry-based approaches. About 60 percent of those used stable isotope labeling methods such as ICPL, iTRAQ and stable oxygen isotope labeling. The remainder used label-free approaches either spectral counting or an ion current-based method.
Members of the PRG calculated a "percentage error of ratio" for each pair of proteins analyzed. The percentage error was equal to the expected ratio minus the observed ratio, divided by the expected ratio.
Of the eight pairs of proteins analyzed, the protein that was the hardest to quantify relatively was glycogen phosphorylase, which was present at a ratio of 1:76. Most laboratories had a relatively high percentage of error ratio for that protein, with the exception of laboratories that used 2D differential radioactivity gels, and label-free mass spec quantitation methods.
In general, most laboratories were able to correctly identify the eight proteins, and to quantify them. Most labs also got good results for proteins that were present at 1:1 ratios.
According to the PRG's analysis, error rates generally went down if a laboratory repeated its quantitation experiments more than once. However, for label-free mass spec analysis, error rates benefited little from repeated analysis.
"2D DIGE and differential radiation did really well. That makes me say that there's a good reason to stay away from non-multiplexed gel methods. Multiplexing is required."
With electrophoresis-based methods, multiplexed methods, including DIGE and 2D differential radiation gels, performed better than non-multiplexed methods such as 2D fluorescence and Coomassie-stained gels.
"2D DIGE and differential radiation did really well. That makes me say that there's a good reason to stay away from non-multiplexed gel methods," said Tracy Andacht, the manager of a proteomics facility at the University of Georgia. "Multiplexing is required."
Turck noted in his concluding remarks that there is no consensus on how to report statistical confidence for quantitative data.
"The acceptable limits of error depend on the analytes and the design of the experiment," he said.
Turck said that he had been using iTRAQ and Coomassie-stained 2D gels to do quantitative analysis in his laboratory, but that he is considering changing his methods given the results of the PRG study.
"Based on these results, I would look into the label-free methods," he said.
When asked why the PRG did not include proteins with closer relative quantities, such as 1:1.2 or 1:1.5 ratio proteins, Turck said the study group members did not want to make their first quantitation study too difficult.
"I would think [those ratios] would also be pretty challenging, and we didn't want to make it too difficult the first time around," he said.
The PRG will probably conduct a follow-up study on quantitation this year, Turck said.
Results from this year's study will soon be posted on the PRG's website at www.abrf.org/prg. A written report of the study will also be published later this year in the Journal of Biomolecular Techniques, which is the official journal of the ABRF.
Tien-Shun Lee ([email protected])