Download Accurate Peptide Fragment Mass Analysis: Multiplexed

Accurate Peptide Fragment Mass Analysis: Multiplexed Peptide Identification and Quantification
Chad R. Weisbrod, Michael R. Hoopmann, Jimmy K. Eng, Tahmina Baker, James E. Bruce
Department of Genome Sciences, University of Washington, Seattle, Washington 98109, United States
Overview
Results
Data independent acquisition technique and bioinformatics
approach for peptide identification and quantification
Identification independent of precursor mass information
Quantification based upon MS/MS fragment ion intensities
Data acquired using novel dual linear RF ion trap FT-ICR MS
Methods
FASTA
Sequence
database
The effect of match count on the FT-ARM analysis of the yeast
peptide LGANAILGVSLAASR. (mass accuracy = ±20 ppm)
Quantification comparison between FT-ICR acquired data processed with
FT-ARM(Left) and triple quadrupole acquired data and processed with
Thermo Xcaliber(Right) of BSA spiked into yeast lysate digest.
Correlational analysis between fragments observed in a DIA LCMS/MS experiment and input data
in a data independent
acquisition is performed. Data is filtered prior to correlation based
upon fragment ion mass accuracy and number of coincident
fragment ion matches (match count).
40
20
2
3
4
5
6
Log10(molecules/cell)
FT-ARM analysis of the yeast peptide LGANAILGVSLAASR
(match count = 8, mass accuracy = ±3 ppm).
No
precursor mass information was utilized in FT-ARM to form
the identification.
Identifications from yeast lysate digest
Unique proteins identified mapped
back to Ghaemmaghami et al.1
yeast protein expression values.
Ion accummulation times between the
LTQ-FT and the Velos-FT.
Conclusions
Measurements that are independent of precursor mass
enable identification and quantitation of modified peptides,
even with unknown or unexpected modifications.This strategy
requires that the modified peptide shares significant overlap
in fragmention to its unmodified form. Above FT-ARM was
utilized to measure phosphorylated angiotensin II using the
unmodified hypothetical spectrum. (Top = TIC, Bottom = FTARM score chromatogram)
Mass spectrometry experiments used for comparison of
conventional bottom-up approach to FT-ARM .
…
Peptide
spectral library
60
80
Velos-FT
LTQ-FT
FT All Reaction Monitoring (FT-ARM)
FT-ARM has been developed to include a GUI.
The GUI enables queuing of multiple analyses
and to perform comparative analysis on multiple
datasets.
DTF and CID fragmentation
patterns of angiotensin I.
Dual trap fragmentation (DTF) is a
method to induce higher energy
fragments in a dual linear ion trap
Velos-FT instrument diagram
0
To date, the most popular method for peptide and protein identification using
mass spectrometry is bottom-up or shotgun proteomics coupled with data
dependent acquisition (DDA) mass spectrometry. Multiplexed fragmentation,
or simultaneous isolation and fragmentation of multiple peptides, has been
developed as an alternative to bottom-up DDA to increase peptide
identification rates. Multiplexed peptide identification from data independent
acquisition (DIA) presents major challenges to modern proteomics, however,
provides the opportunity for parallelizing an otherwise serial peptide
measurement.
Parallelizing peptide identification and quantification may
represent a significant future direction of mass spectrometry based
proteomics, just as parallelized genome sequencing machines have advanced
the field of genomics.
The effect of mass accuracy on the FT-ARM analysis of the
yeast peptide LGANAILGVSLAASR. (match count = 3)
Number of Unique Proteins
Introduction
Novel dual linear RF ion trap FT-ICR mass
spectrometer: “the Velos-FT”
The
overlap
in
identified
peptides
between
the
two
approaches
is
approximately
40%.
FT-ARM complements
data
dependent
analysis.
Histogram of peptides identified in each 100 Da window FTARM DIA experiment compared directly with conventional
bottom-up proteomics approach. Inset is a histogram from
a separate FT-ARM experiment with narrower (12 Da) DIA
windows.
Complementarity of FT-ARM results are in part due to better
identification of co-isolated peptides.
FT-ARM – A novel DIA analysis method which relies on high mass
measurement accuracy and peptide fragmentation patterns
Coincident quantification and identification
Identification rate comparable to conventional DDA
Quantification in complex mixtures to low femtomole levels
FT-ARM will complement the development of an array based FT-ICR mass
spectrometer
Novel Velos-FT mass spectrometer was built and tested
References
1) S. Ghaemmaghami; W. Huh; K. Bower; R. Howson; A. Belle; N. Dephoure; E. K. O'Shea and J. S. Weissman, Global
analysis of protein expression in yeast. Nature 425, 737-741(16 October 2003)
Acknowledgements
This research was supported in part by National Institutes of Health grants
7S10RR025107, 5R01GM086688, 5R01RR023334 and the University of
Washington's Proteomics Resource (UWPR95794). The authors also would like
to thank Dr. Priska D. von Haller for helpful discussion.
For reprints see: http://brucelab.gs.washington.edu/presentations.php