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Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry andProteomics/Metabolomicsusing R and Bioconductor
Laurent [email protected]
Cambridge Centre For ProteomicsUniversity of Cambridge
European Bioinformatics Institute (EBI)
18th November 2010
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Plan
1 Mass SpectrometryMass Spectrometry (MS)SeparationSchematic workflow
2 R/Bioconductor packages
3 Applications and challenges
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Plan
1 Mass SpectrometryMass Spectrometry (MS)SeparationSchematic workflow
2 R/Bioconductor packages
3 Applications and challenges
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Short definition
Mass spectrometry (MS) is an analytical technique thatmeasures the mass-to-charge ratio of charged particles.
Used to study various chemical compounds – peptides (assurrogates for proteins) or metabolites (see xcms talk).
Allows identification and quantification.
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Separation based on analyte physical properties
HP[L|G]C (online) → chromatogram
1D and 2D Gels using Cy-dyes labelled proteins (digeR talk)
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Separation
MS stages
Across retention time. . .
Ionisation source – formation of gas-phase ions (analytes):ESI, MALDI
Mass analyzer – separation of the ions according to their mass(M) to charge (Z) ratio.
Detector – ion current monitoring and amplification (ioncounts)
→ mass spectrum (intensity vs. M/Z)
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Schematic MS(MS) worflow
Precursor iondissociation
Detector
Precursor ions MS1
Fragmented ions
MSMS - MS2
AnalyserSourceSeparation
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Schematic MS(MS) worflow
Precursor iondissociation
Detector
Precursor ions MS1
Fragmented ions
MSMS - MS2
AnalyserSourceSeparation
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
MS1 scan
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Schematic MS(MS) worflow
Precursor iondissociation
Detector
Precursor ions MS1
Fragmented ions
MSMS - MS2
AnalyserSourceSeparation
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Schematic MS(MS) worflow
Precursor iondissociation
Detector
Precursor ions MS1
Fragmented ions
MSMS - MS2
AnalyserSourceSeparation
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
MSMS scans
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
MSMS scans
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
MZ vs. time vs int.
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Identification
MS1 M/Z for the usual suspects in metabolomics.
MS2 spectra are matched against theoretical spectradatabases.
De novo peptide sequencing.
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Mass Spectrometry (MS)SeparationSchematic workflow
Quantification
Using MS1 data over retention time (Bernd’s talk).
In MS2 using spectral counting, assessing abundance basedon protein coverage or using reporter ions (see MSnbase).
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Plan
1 Mass SpectrometryMass Spectrometry (MS)SeparationSchematic workflow
2 R/Bioconductor packages
3 Applications and challenges
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
R/Bioc packages
Precursor iondissociation
Detector
Precursor ions MS1
Fragmented ions
MSMS - MS2
AnalyserSourceSeparation
XCMS (Steffen Neumann)
digeR(Yue Fan)
Label-free MS (Bernd Fischer)
MSnbase (Laurent Gatto)ProtIO (Simon Perkins, Crispin Miller)
MIAPE:MSI(FlorianBreitwieser)
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Plan
1 Mass SpectrometryMass Spectrometry (MS)SeparationSchematic workflow
2 R/Bioconductor packages
3 Applications and challenges
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Applications
Identify peptides/proteins/metabolites.
Relative and absolute quantification (xcms, MSnbase anddigeR talks).
Post-translational modifications (PTM).
Interaction partners (PPI, P-metabolite)
Sub-cellular localisation of proteins/peptides (see pRoloc
talk).
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Some challenges
Dynamic range (6 orders of magnitude in human cells and>10 in serum. . . and no PCR)
Chemical complexity (membrane vs. soluble proteins)
Only some, and preferentially most abundant once, analytesare (1) randomly sampled for MS1 and (2) selected for MS2→ missing data.
Identification is entirely dependent on the quality of theannotation at hand.
Peptide surrogacy: non-unique peptides, how many peptidesper protein, mapping multiple modifications to proteins.
L. Gatto Bioconductor Developer Meeting 2010
Mass SpectrometryR/Bioconductor packages
Applications and challengesThe programme
Session progamme
Metabolomics using xcms (Steffen Neumann)
Label-free differential quantification for proteomics (BerndFischer)
MSMS data with MSnbase (Laurent Gatto)
Protein localisation with pRoloc (Laurent Gatto)
Interfacing proteomics data and R/Bioc with ProtIO (CrispinMiller and Simon Perkins)
DIGE gels with digeR (Yue Fan)
MIAPE:MSI and pep. → prot. (Florian Breitwieser)
Discussion
L. Gatto Bioconductor Developer Meeting 2010