Digging for disease markers of sepsis
fied. Shen used the spectral count—the number of mass spectra produced for a Using a novel combination of techparticular protein—to assess the relaniques, Gary Siuzdak and colleagues tive abundance of each one. He then at Mass Consortium Corp.; Becton, compared the relative concentration Dickinson and Co. (BD); and the Scripps of each protein in a SIRS sample to that Research Institute identified 10 potenof the same protein in a sepsis sample. tial biomarkers that distinguished the This comparison showed that 103 proserum of patients with sepsis from that teins were either more concentrated of other very sick patients. They report or less concentrated in some sepsis their findings in this issue of JPR (pp samples than in some SIRS samples. 3154–3160). This disease “turned out to Using the database for annotation, be one of the more difficult problems we visualization, and integrated discovery could have gone after, because sepsis (known as DAVID), William Nussbaucan result from many different sources,” mer from BD grouped the 103 proteins says Siuzdak. Sepsis, which kills into biologically meaningful >200,000 Americans every year, subsets. The complement and develops when the bloodstream coagulation pathways were the is overwhelmed by toxin-producbest-represented systems. ing microorganisms. When all the sepsis samples The researchers’ initial atwere compared with all the SIRS tempt to find sepsis markers samples, seven proteins were simply showed that sick people found to be consistently updiffer from healthy people, and regulated and three consistently they detected no differences in down-regulated. Acute-phase easily identifiable serum proproteins, complement proteins, teins that might indicate early coagulation proteins, and lipid sepsis. Then, scientists at BD transporters were all represented. performed a longitudinal study The up-regulated proteins were of intensive care unit (ICU) complement component C4 and patients who did and did not dethe precursors to C-reactive provelop the disorder. As expected, tein, plasminogen, apolipoprotein In the blood. To find biomarkers for sepsis, researchers the typical high-abundance A-II, plasma protease C1 inhibitor, immunodepleted high-abundance proteins from serum, a serum proteins dominated the transthyretin, and serum amyloid component of blood, and analyzed the remaining proteins by mass spectral data, making it P-component. Among the promultidimensional LC and tandem MS. impossible to obtain high-qualteins that were consistently less ity quantitative information on abundant in the sepsis samples the less-abundant proteins. each of the SCX runs, ultimately allowthan in the SIRS samples were the At about this time (2003), the muling us to have a manageable number of precursors to apolipoprotein A-I, antitiple affinity removal system (MARS) peptides for the final reversed-phase thrombin-III, and serum amyloid A-4. was unveiled. These immunodepletion runs,” Siuzdak explains. “There were clearly more proteins that columns, consisting of antibody-coated The resulting fractions were anawe could have listed, but those were the beads, cleanse serum of its six most lyzed by MS/MS, which generated ~10 most significant ones,” says Siuzdak. abundant proteins: albumin, transmillion spectra. Only 2% of the spectra MS is already widely used in neonatal ferrin, haptoglobin, antitrypsin, and represented the 6 high-abundance proscreening to diagnose inborn errors immunoglobulins G and A. The Scripps teins, compared with 59% from those of metabolism. “This work further researchers applied the BD samples to without immunodepletion. demonstrates that [MS] can also be a a MARS column. The serum came from Having successfully obtained so powerful research tool for untargeted 25 ICU patients with systemic inflammany spectra from low-abundance screening of disease-related proteins,” matory response syndrome (SIRS) and proteins, the researchers’ next chalSiuzdak says. 25 with sepsis, in which the inflammalenge was data analysis. “The difference Shen says their approach is likely to tory response is associated with a docubetween our paper and previous papers be even more powerful than demonmented infection. Sepsis is more deadly is that we acquired a huge amount of strated in the paper because mass specthan SIRS and, therefore, requires more data using the MS instrument that trometers have improved dramatically aggressive medical attention. “For a was available at that time,” says Mass in the 2 years since the study ended. physician to be able to identify [sepsis] Consortium Corp.’s Zhouxin Shen, who “Our method is universal,” he says. “We at an early stage would be very valugenerated and analyzed the data. Using picked the most difficult disease but able,” Siuzdak says. a proprietary proteomics analysis platstill found potential biomarkers from The immunodepletion step removed form, Shen found 2810 proteins in the small differences.” 85% of the protein from the samples. samples. Of those, 484 could be identi—Linda Sage PHOTODISC
The remaining proteins were digested with trypsin, which resulted in ~1 million peptides. Instead of the typical 2D LC separation, a 3D approach was used to fractionate these peptides. First, the digests were passed through a reversedphase column from which peptides were eluted with organic solvent and fractionated by hydrophobicity. The fractions were then run on a strongcation exchange (SCX) column, which separates peptides by ionic strength. The third step was another reversedphase column. “Doing the initial reversed-phase fractionation simplified
Journal of Proteome Research • Vol. 5, No. 11, 2006 2885
