Post-digestion trypsin-catalyzed 16O/18O labeling coupled to the AMT tag approach has been applied for global quantitative proteome analysis of human plasma following LPS administration. The results demonstrate that the proteome-wide precise quantitation of different physiological conditions can be achieved using this combined strategy, allowing confident identification of a set of proteins having significant changes in concentration. The coupling of the AMT tag approach with automated data analysis provided the basis for high throughput proteomic measurements without the need for routine LC-MS/MS measurements. The overall efficiency, quantitative precision, and throughput of this strategy make it suitable for comparative proteome analysis of large numbers of clinical samples for biomarker discovery.
There are several advantageous features of the trypsin-catalyzed 16
O labeling, including: (1) The post-digestion labeling methodology incorporates 2 atoms of 18
O in essentially all tryptic peptides, providing the framework for accurate quantitation; (2) the enzyme catalyzed approach can be applied to label tryptic peptide samples from various biological sources, including tissues, cell lysates, and biological fluids; and (3) the 16
O labeling can be easily coupled to peptide-specific enrichment methods such as cysteinyl-peptide enrichment,11
or to peptide-fractionation techniques such as SCX to improve overall proteome coverage of the analysis. However, one of the concerns related 18
O labeling is that the 18
O-labeled peptides may exchange back to 16
O, albeit at a low rate of exchange, that will potentially decrease the overall efficiency of the stable isotope labeling and quantitation. Instead of using cysteine alkylation of trypsin under denaturing conditions as suggested by a previous report13
, we found that the residual trypsin activity following digestion can be effectively quenched by boiling the samples for 10 min and then immediately placing the samples on ice. In the post-digestion labeling methodology, the use of immobilized trypsin allows the enzyme to be completely removed following the labeling step. Using this protocol we have not observed any evidence of oxygen back-exchange even after the labeled samples were stored for several months in normal water (data not shown).
We have presented an initial demonstration of the comparative analysis of human plasma without depletion of any major proteins, quantifying a total of 429 non-redundant plasma proteins from clinical human plasma samples. The dynamic range of concentration for proteins present in human plasma is expected to be >1010
, which presents a significant challenge for detecting low abundance proteins. While the results from this study demonstrate the effectiveness of identifying proteomic changes between different clinical plasma samples, the limitation in overall detectable dynamic range has resulted in the quantification of proteins that are primarily considered as having a medium to high abundance levels. Thus, the number of proteins observed with significant changes in concentration in plasma is limited by the coverage or low signal levels of lower abundance proteins due to the presence of a limited set of very high abundance proteins. To improve the overall dynamic range of detection, new depletion strategies for removing high abundance proteins2
and novel enrichment methods for enriching specific subsets of peptides such as cysteine-containing peptides11, 33
and N-glycosylated peptides34
are essential and can be applied in combination with the present approach. The application of such depletion or enrichment methodologies is highly promising for extending the present quantitative analysis approach to much larger numbers of low abundance proteins in plasma.
The extension of the reported method to a study of the plasma time-dependent acute phase response following LPS administration is presently in progress that aims to provide an extended dynamic range proteome survey of potential mediators in inflammatory response that may contribute significantly to our understanding of systemic inflammation and sepsis syndrome. With the increased dynamic range of detection resulting from the additional application of depletion and/or enrichment strategies to proteomic samples, we anticipate broad utilization of this quantitative approach in clinical plasma/serum proteomics.