A next generation label-free quantitation strategy for analyzing
complex peptide mixtures
Paulo C Carvalho*1,2, Xuemei Han*1, Tao Xu1, Daniel Cociorva1, Maria da Gloria
da Costa Carvalho3, Valmir C Barbosa2, and John R Yates, III1
1. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, USA
2. Systems Engineering and Computer Science Program, Federal University of Rio de Janeiro, Brazil
3. Biophysics Institute, Federal University of Rio de Janeiro, Brazil
Label-free approaches (e.g., spectral counting) for the quantitation of complex peptide
mixtures have gained popularity because of their low cost, reasonable accuracy, and
simplicity. However, they still fall behind in accuracy from those that measure abundance
by comparing peptides to an internal, chemically identical standard enriched with a heavy
stable isotope. Nevertheless, the latter are expensive and laborious. Here we describe a
novel approach for shotgun proteomic data acquisition, termed extended data-independent
acquisition (XDIA), which greatly reduces the gap between these technologies. XDIA
combines high-resolution Orbitrap survey scans with multiplexed fragmentation data
acquired using an ETD/CID double play. The data are then post-processed by our new
software, the XDIA Processor, which enables for protein identification. A schematic of
this process is shown in Figure 1.
We acquired spectral counts on a yeast lysate digested with Lys-C using XDIA and
compared the result to the one obtained by the widely adopted data-dependent acquisition
(DDA) using ETcaD. Both identified roughly the same number of proteins; however,
XDIA was more reproducible, identified ~250% additional spectra, and ~30% more
unique peptides per run. Boosting the number of identified spectra significantly improves
the quantitation statistics confidence; increasing identified peptides improves on coverage,
making XDIA a key contribution.
Figure 1 – Data-Dependent versus
Extended Data-Independent Acquisition.
In the DDA approach, an MS1 is acquired
so precursor ions can be selected for
posterior analysis by MS2. In the XDIA
approach, a wide precursor window that can
include multiple peptide ions is selected for
fragmentation. The precursors and their
charge states are detected by the XDIA
processor and are included in the final MS2
file.
Acknowledgments: CNPq, a FAPERJ BBP
grant, and NIH P41 RR011823, R01
MH067880 for financial support.