Transcript The QSTAR XL (2003)

ASAS
Q-TOF Mass Spectrometers
Martin Middleditch
ASAS Mass Spectrometry Manager
ASAS Q-TOF Mass Spectrometers
The QSTAR XL (2003)
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~$70-150 per sample
Infusion or capillary-scale 1D or 2D LC-MS/MS.
To be made available for direct access by trained users in 2015
Resolution ~10,000, mass accuracy ~10-50ppm, ~1MS/MS per sec.
Suitable for intact protein MW determinations (+/- 0.003%), identification
of proteins from gel bands, biofluids, solubilised tissue etc, and quantitative
comparisons of proteomes using iTRAQ labelling.
The TripleTOF 6600 (2014)
~$120-480 per sample
Equipped for nanoscale infusion or nanoLC-MS/MS workflows.
Resolution ~35-50,000, mass accuracy ~2ppm, up to 100 MS/MS per sec.
High speed acquisition rate permits new workflows such as SWATH.
Best choice for identifying thousands of proteins per sample, relative
quantitation of complex proteomes, and validating any changes in peptide
abundance by scheduled MRM workflows.
• By mid-July, will also have a SelexION Differential Mobility Separation
module to separate analytes by their mobility in high and low electric fields.
• Future directions: Comprehensive phosphoproteomics, lipidomics
(Lipidyzer+SelexION), unknown/metabolite identification.
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Intact Protein MW determinations
Human Alzheimers Brain on TripleTOF
Base Peak Chromatogram
Data searched against all
human sequences using
ProteinPilot v5.0
MS/MS trace
24,230 MS/MS spectra
13,672 peptides identified
1,619 proteins identified
1min window
of MS/MS trace
MS/MS Spectral Matching
Relative Quantitation Using iTRAQ Tagging
SWATH Analysis
1. Download or create (by a thorough
IDA analysis of a pooled sample), an
Ion Library for your species, containing
the identification data for 1000s of
proteins.
2. Generate SWATH datasets for each
sample
by
sequentially
acquiring
MS/MS data on small bins (e.g. 25Da)
of precursors across the entire mass
range of interest in every cycle (~2-5
seconds).
3. Post-acquisition, mine the data from
each bin using very narrow extraction
windows for the predetermined peptide
fragment ion patterns in the ion library.
Aebersold et al, 2014
4. Concurrent elution of fragment ions
at the expected normalised RT, with
the expected relative intensities to one
another constitutes the assay data for
quantitative comparisons of peptides.
SWATH-MS for Proteomics
“…a generic large-scale human assay library to
support protein quantification by SWATH-MS.
It consists of 1,164,312 transitions identifying
139,449 proteotypic peptides and 10,316 proteins.
Generated by combining the results from 331
measurements of fractions from different cell
lines, tissue and affinity enriched protein samples.
The assays consist of precursor and fragment ion
m/z, normalized RT and relative ion intensities…”
SWATH-MS Proteomic Dataset
Aebersold et al, 2013
Illumina’s BaseSpace OneOmics Proteomic Tools
Use Cloud Computing to process
large SWATH-MS datasets.
Protein Areas
Link Proteomic results to Genomic
datasets from the same samples.
Peptide Areas
Transition Areas
Can visualise quantitative
comparisons at the Protein,
Peptide and Transition levels
SelexION Differential Mobility Interface
Differential Mobility Cell
Modified Curtain Plate
ESI source
TripleTOF
SelexION Differential Mobility Separation
ESI source
Sample Ions
TripleTOF
Abdi, 2013
Lipid Class Separation Using SelexION
MRM scan of 6 phospholipid standards
using SelexION with a COV ramp
PI
PG
PS
PE
PC
PA
Compensation Voltage
Can resolve similar molecules
by exploiting differences in their
mobility when subjected to
alternating high and low electric
fields.
Different molecules will have
different optimal Compensation
Voltage (COV) values for their
transmission into the MS.
Useful for structural isomers
that can be difficult to resolve
chromatographically, and are
impossible to resolve by MS
alone due to their identical
formula.
Abdi, 2013
SelexION DMS resolves both Positional and
R and S Isomers of Hydroxy-eicosatetrenoate
Abdi, 2013
Thank you for your attention