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of DDA and DIA allows for
he MS1 peaks for two different
tra for the top 4 transition ions
Given that these two peaks are
be difficult to confidently
tation without added MS/MS data
is the correct precursor for
ed in D.
The primary challenge to global protein characterization is attributed to data processing.
Reliance on spectral libraries not only helps to determine the peptide list used for post-
acquisition data processing, but also provides robust scoring metrics to significantly
decrease manual peak integration. Basic DIA events using 25 Da precursor isolation
windows result in greater numbers of nonsymmetrical, overlaid product ion XIC traces that
introduce errors in automated peak picking routines. By decoupling the HR/AM MS and 5
Da DIA data, each acquisition event can leverage high-resolution in both MS and DIA
spectra to reduce target ion signal from background. This decreases the need for manual
post-acquisition processing. In addition, by modeling sequencing strategies around DDA
events (
e.g.
narrow precursor isolation and only one correctly matched product ion
spectrum needed for confident sequencing) the selectivity and sensitivity of each 5 Da DIA
window can be increased with longer maximum ion fill times and higher resolution settings
than previously reported for basic DIA experiments. This combination increases the
robustness of automated data processing and provides larger lists of differentially abundant
peptides that can be globally evaluated with ROC analyses for classification value. The
results of the ROC analysis can provide a starting point for translation to targeted, high-
throughput methods.
DA or DIA alone.
sitivity and selectivity, since the
qualitative and quantitative
tion of MS1 quantification due to
uired within the narrow windows.
peed and higher resolution
n Accurate Mass (HR/AM)
ble robust complex sample
ethods. Decoupling
the high-resolution capabilities
meter. The combination of data
ide determination and
ks that are chromatographically
topic distribution profiles. The
indow acquisitions allows for
ral matching of the specific 5 Da
ach peak, clearly identifies the
sed on the presence and
ntry,
Conclusion
We have created a high quality murine heart tissue spectral library with over
5900 protein/19,900 peptide targets at FDR 1%.
Using the pSMART workflow, we can quantify at MS1 level with high confidence
in label-free discovery experiments using MS/MS spectra for confirmation of
each quantified precursor ion area.
The pSMART workflow coupled to HR/AM MS instrumentation provides
increased protein assignments with lower FDR compared to standard DDA and
DIA methods
1-3
.
References
1. Prakash, A., Peterman, S., Ahmad, S., Sarracino, D., Frewen, B., Vogelsang, M.,
Byram, G., Krastins, B., Vadali, G., Lopez, M. Hybrid data acquisition and
processing strategies with increased throughput and selectivity. Journal of
Proteome Research (submitted).
2. Egertson, J.D., Kuehn, A., Merrihew, G. E., Bateman, N. W., MacLean, B. X.,
Ting, Y. S.,Canterbury, J. D., Marsh, D. M., Kellmann, M., Zabrouskov, V., Wu, C.
C., MacCoss, M. J., Multiplexed MS/MS for improved data-independent
acquisition. Nature Methods, 2013. 10(8): p. 744-748.
3. Gillet, L.C., Navarro, P., Tate, S., Rost, H., Selevesk, N., Reiter, L., Bonner, R.,
Aebersold, R., Targeted data extraction of the MS/MS spectra generated by
data-independent acquisition: a new concept for consistent and accurate
proteome analysis. Molecular & Cellular Proteomics, 2012. 11: p. 1-17.