Quantitative proteomics analysis of maternal plasma in Down syndrome pregnancies using isobaric tagging reagent (iTRAQ)

Abstract

Currently no specific biomarkers exist for the screening of pregnancies at risk for Down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in betaHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers.

Figure 1

Workflow for quantitative proteomics using iTRAQ reagent. Equal amounts of plasma protein (100 μg) from control and DS (n = 6) were pooled separately and duplicated, controls were labeled with 114 and 116 iTRAQ label and DS was labeled with 115 and 117 iTRAQ label. The labeled samples were pooled and were subjected to a strong cation exchange chromatography to remove the excess label. Afterwards LC-MALDI MS/MS was performed for protein identification and quantification.

Figure 2

Components of the spectrum illustrated are (i) MSMS spectrum of the precursor ([M + H] +, m/z 1527.7 Da). (ii) low-mass region showing the reporter ions used for quantitation. The peptide is labeled by isobaric tags at both the N terminus and C-terminal lysine side chain. The precursor ion and all the fragment ions therefore contain all four members of the tag set, but remain isobaric. The MSMS spectrum was obtained from the singly charged [M + H] + peptide using a 4800 MALDI TOF-TOF analyzer.

Figure 3

Frequency distribution (bars) from both DS and control replicates across different ranges of variation. The cumulative percentage (lines) is defined as the cumulative number of proteins falling within the defined variation range against the total number of protein.

Figure 4

Number of plasma protein identified using iTRAQ reagent. In total, 235 proteins were identified. Shown above is the classification of these protein in different category based on molecular function.

Figure 5

PANTHER analysis for pathway. In total, 28 proteins were identified as elevated and 22 proteins were under expressed. Shown above is the different signaling pathways hits by these protein.