BEIJING — During one and a half days of workshops here to discuss results of the pilot phase of the Human Proteome Organization’s Plasma Protoeme Project, plasma researchers grappled to decide which techniques should be standardized to come up with harmonious data across laboratories, and how some techniques such as mass spectrometry can be standardized.
“It’s clear that pre-analytical factors such as specimen type, collection and processing, storage and transportation do affect the final result,” said Daniel Chan, the director of the Biomarker Discovery Center at Johns Hopkins University, and the chair of the specimen collection and specimen handling subcommittee for the HPPP.
Founded in 2002, the HPPP lists as long-term goals the need to comprehensively analyze proteins in plasma and serum; to characterize variation within individuals arising from physiological, pharmacological, and pathological processes; and to explore variation across individuals within populations and across populations.
In 2003, the pilot phase of the HPPP began with the preparation and distribution of reference specimens to 31 laboratories. The purpose of analyzing reference samples across labs was to standardize procedures for analyzing plasma and serum.
According to Gil Omenn, the chairman of the HPPP and a professor at the University of Michigan in Ann Arbor, researchers reported 9,506 different proteins across the 31 labs after analyzing the reference plasma and serum samples. Of those, more than 3,020 were reported in at least two specimens or two labs.
Differences in data sets were attributed in part to differences in specimen handling; whether most abundant proteins were depleted before analysis; or whether there were differences in equipment used, such as mass spectrometers and HPLC columns.
The issue of whether to remove the most abundant proteins before analysis stems from the fact that 10 proteins make up 90 percent of the protein mass in serum, and 22 proteins make up 99 percent. If the most abundant proteins, such as albumin, are not removed using affinity columns such as those manufactured by Agilent and Applied Biosystems, they appear as a large smear that can mask other, less abundant proteins.
While many researchers agreed that depleting most abundant proteins is a wise step that will enable researchers to identify more proteins, Richard Simpson, a professor at the Ludwig Institute in Melbourne, Australia, and the chair of the HUPO New Technology and Resource Committee, said he is not convinced that removing abundant proteins allows for lower abundance proteins to be identified.
“I believe the jury is still out,” said Simpson. “Are we actually probing lower [abundance proteins], or are we seeing the same proteins with a higher level of confidence?”
To analyze whether low-abundance proteins are being stripped away along with high-abundance proteins when they are removed, some researchers treated albumin as a “molecular sponge” that they analyzed after stripping for the presence of other proteins.
Other issues discussed during HPPP workshops included whether mass-spectrometry techniques should be standardized, and the reliability of manual validation of mass spectrometry data.
Catherine Fenselau, the president of US HUPO and a professor of biochemistry at the University of Maryland, said that different mass spectrometry techniques, such as electrospray ionization, nanospray ionization, and MALDI, inherently yield different peptides, and that it would be unwise to try to standardize the various techniques.
“Mass spectrometers are expensive, and they’re often chosen based on other criteria besides the HPPP,” said Fenselau. “It would be a bad idea to try to harmonize the instrumentation used in HPPP. I don’t think the HPPP was designed to do an instrumentation comparison.”
In general researchers agreed that LCMS-MS is the best way to analyze complex mixtures of proteins, such as those present in plasma.
In terms of validation of new plasma and serum proteins, Omenn questioned the reliability of laboratory members putting a check mark next to a protein saying that it has been “manually validated.” He cautioned that like pathologists and radiologists in medicine who often come up with different diagnoses, laboratory researchers are likely to come up with different results when given the task of validating a new protein.
Omenn suggested that, as with radiology, some sort of scoring system might be devised that compares analyses from several different protein validators.
“There are elaborate scoring systems that are routine in radiology where more than one radiologist evaluates, and results must be within a range of scores,” Omenn explained. “The same thing could be done in this field.”
One of the most exciting results that came out of the pilot phase of the HPPP study was the identification of 94 proteins that come from novel genes which had no known product before, said Omenn.
“That’s a bonus,” said Omenn.
Abstracts of studies done by all 31 pilot-phase HPPP laboratories were published in a special edition of the journal Molecular and Cellular Proteomics.
The next step after this pilot phase will be to further clarify preliminary results and to organize strategies for large-scale studies to identify plasma and serum biomarkers, said Omenn.
— TSL