Validating biomarkers in CSF
1022.45
1028.47
2 pmol of 14 reference peptides that to facilitate the integration and analysis Because Jing Zhang finds the search for matched proteins of interest. Each of the data generated from MALDI TOFneurodegenerative markers a “tough, peptide contained one 15N atom and TOF, which was not a trivial job,” Zhang tough job,” he and colleagues at the five or six 13C atoms. The peptides in the says. University of Washington School of spiked fractions were further separated In this proof-of-principle study, the Medicine and six other institutions with reversed-phase LC and spotted researchers validated three biomarkers have developed a platform for the valionto MALDI plates. The next step was unique to patients with Alzheimer’s dation of biomarkers in cerebrospinal a quick MALDI TOF-TOF MS screen to disease (AD) or late versus early stages fluid (CSF) and report their results in look for targeted peptides within the of Parkinson’s disease (PD): neurexin-1, JPR (2008, 7, 720–730). The platform, mass search window that showed the α-1-acid glycoprotein, and β-2-glycobased on LC/MALDI-TOF-TOF MS, correct mass. “But that doesn’t mean protein 1. involves reference peptides labeled that all of the precursors selected based The total abundance of neurexin-1 with stable isotopes for targeted quan on their masses are the peptides that (all three isomers), as determined by a titative analysis. The advantages, we are looking for, because different representative peptide, was higher in according to Zhang, are multiplexing, peptides can have very similar masses, middle-aged controls than in the older robustness, high specificage-matched controls, suggestity, the potential for high ing that levels of this cell-surthroughput, and the abilface adhesion protein decline ity to detect proteins in with age, even in healthy subATVVYQGER complex samples without jects. Levels were even lower in antibodies. Moreover, the the AD and late PD subjects. dissociation of sample The level of α-1-acid glycoReference preparation from MS protein was strikingly higher (rather than combining the in the AD samples than in the two, as in ESI MS) makes controls. Because this protein Native it possible to return to any is involved in the inflammaMALDI spot for further tory response during the acutestudy. Currently, Zhang’s phase reaction, the researchers 1017.0 1020.8 1024.6 1028.4 1032.2 1036.0 group is using the platform suggested that some or all of Mass (m/z) to verify biomarkers for the AD subjects might have exParkinson’s, Alzheimer’s, perienced neuroinflammation. It’s in the spectrum. MS data for a peptide with the correct mass and other neurodegeneraβ-2-glycoprotein 1 also (1022.45) to correspond to isotope-labeled ATVVYQGER (1028.47), a tive diseases. was up-regulated in the AD peptide in β-2-glycoprotein 1. “The validation of CSF samples. Because this antigen markers discovered by for antiphospholipid antibodproteomics is a major ies has not yet been linked to challenge and critically important for especially in a complex sample,” says AD, the researchers will try to validate biomarker development,” Zhang says. coinvestigator Sheng Pan, who is also at it, along with other recently discovered “Because of the many caveats during the University of Washington. potential neurodegenerative biomarkthe discovery phase, most results are To identify the targeted peptide pairs ers, in CSF from a larger patient cohort. not really reproducible, especially (a reference peptide and the native pepAlthough they identified proteins in among different labs.” tide with the correct mass difference), this study, the researchers point out that Human CSF contains thousands of the researchers performed MS/MS on their platform would also allow pepproteins with concentrations ranging selected precursor ions and searched tides to be used as markers of disease. over 9 orders of magnitude. Antibodies the resulting data against a protein da“If you have a peptide that is unique against only a few of those proteins are tabase to identify the correct sequences. to AD, you don’t need to know what commercially available. When Zhang’s “Complementary identification using protein it represents,” Zhang says. “This group began its proteomic analyses accurate mass, isotope pattern, and MS/ issue is important because proteins in4 years ago, just 100 CSF proteins had MS analysis provides high confidence ferred by peptide sequence may not be been identified. The researchers have on targeted identification of candidate correct due to the current incomplete boosted that number to >2000. peptides,” says Pan. human protein database.” To validate biomarkers with the new To quantify peptide levels, the group The researchers also stress the need platform, the group digests the proconstructed a calibration curve, in to use a variety of techniques to find teins in CSF samples with trypsin and which the y axis indicates the peak biomarkers of disease. “The LC/MALDIfractionates the resulting peptides with intensity ratio of a known amount of naTOF-TOF-based platform provides a strong-cation exchange chromatogrative peptide (represented by unlabeled complementary approach for the verifiphy. Then, they spike each fraction with synthetic peptide) to the corresponding cation and validation phase, especially a mixture of synthetic, labeled reference spiked-in reference peptide, and the x for proteins for which no specific antipeptides. axis indicates the concentration of “nabodies have been developed,” Pan says. In this study, the researchers added tive” peptide. “Software was developed —Linda Sage
Journal of Proteome Research • Vol. 7, No. 2, 2008 483
