Gentle ionization mass spectrometry as universal research tool in life science.
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Transcript of Gentle ionization mass spectrometry as universal research tool in life science.
Gentle ionization mass spectrometry as universalresearch tool in life science
Mass spectrometry: generation and detection of ions
Two gentle ionization techniques permit analysis of biomaromolecules
MALDI ESI
Matrix assisted laser desorption ionization Electrospray ionization
López Neyra EEZ
MALDI MALDI-TOF (time of flight detector)
MALDI
MALDI sample plate
- Higher precision (de novo identification)- Higher mass range (up to 500 kDa)- Higher salt tolerance- Easy to automatize
- 0-500 Da mass range impossible- Impossible to connect LC- Risk of breaking labile covalent interactions
- does not break covalent interactions, but breaks the very large majority of non-covalent interactions (protein unfolding)
- Solvent must be volatile: water, organic solvents, ammonium carbonate/acetate
- Positive mode (addition of 0.1 % formic, acetic or trifluoroacetic acid by operator)
- Negative mode (addition of ammonia)
- Ionisible: proteins, peptides, sugars, nucleotides, ADN, ARN, fatty acids, low molecular weight compounds, metabolites (most compounds in life science)
- Non ionisible compounds: hydrocarbons
ESI
The principle
HPLC
Syringe pump
Information on the molecular weight of the entire compound (parent ion)Information on the massess of fragments (daughter ions)
Parent Daughter
Use of ESI-MS in life science
Syringe pump
m/z ratios
Not masses but mass/chage ratiosare measured
Mass spectrum of two peptides Mass spectrum of hemoglobin
Micro-heterogenetiy of molecules
Krell et al. (1995) Acta Cryst. D53, 612.
Heterogeneity:Chicken ovalbumin
59 diferent forms
Untreateddeglycosylated
dephosphorylateddeglycosylatedanddephosphorylated
Yang et al. (2013) Anal. Chem. ;85,12037
Analysis ofoligonucleotides
Reyzer et al. (2001) NAR 29:E103-3.
Negative mode
Study of phospholipids
Brooks et al. (2002) J. Exp. Botany 205, 3989
Nag et al. (2004) Am J Physiol lung Cell Mol Physiol 287:L1145-53
Mass spectrometry and quantification
SA: sebacic acid
TA: terphthalic acid
DDA: 1,12-dodecanedioic acid
Rizzarelli et al. (2011) Anal. Chem. 83, 654
Method to quantify SA and TA in complex mixtures
Mass spectrometry & simpleKinetics: stability of phosphorylated
phosphoglycerate mutase
Question: stability of phosphorylated PGMKrell et al. (1998) J. Peptide Res. 51, 201
Mass spectrometry & simpleKinetics: stability of phosphorylated
phosphoglycerate mutase
The experimental set-up
Phosphorylate PGMwith 2,3 bisphosphoglycerate
Separate phosphorylated PGNfrom excess 2,3 bisphosphoglycerate
Phosphorylated PGMInject into spectrometer at regular intervals
Krell et al. (1998) J. Peptide Res. 51, 201
Mass spectrometry & simplekinetics
Phosphorylation half-life: 35 minutes
T=0
T=18 min
Krell et al. (1998) J. Peptide Res. 51, 201
Syringe pump
Parent Daughter
The power of fragmentation
The power of fragmentation:glycosylation
GlycopeptideParent ions[M+3H]
Daughther ions of932
Damen et al. (2009) J.Am. Soc. Mass Spec. 20, 2021
The power of fragmentation:
peptides
PeptideParentions
afterfragmentation
The power of fragmentation:
compounds
Paiva-Silva et al. (2006) PNAS 103, 8030
ESI-MS fragmentation spectrumof heme
Identification of heme type
HPLC – MS/MS
HPLC
Parent Daughter
Separation of compounds (Lourdes)Separation of peptides (peptide mapping)
HPLC –Ms/Ms for peptide mapping
HPLC –Ms/Ms to identify post-translational modification sites
Peptide map of deglycosylated protein
Peptide map ofglycosylated protein
HPLC-MS/MS: The limits
Tryptic digest of albumin (67 kDa)Peptides identified by ms are numbered
HPLC MS to identifynon-covalent binding sites
Krell et al. (1998) J. Peptide Res. 51, 201
Example: identification of the substrate binding site in shikimate dehydrogenase
Enzyme is rapidly inactivated by trinitrobenzene sulfonate, a lysine specific reagent
Mass increase by 211 Da
Identification of active siteresidues by HPLC peptide mapping
Spectrum of full-length protein treated with TNBS
3 lysine residues modified
Spectrum of full-length protein treated with TNBS in the presence of substrate
1 lysine residue modified
Micro-demanda
- Menos de un día de trabajo
- Análisis sobre la marcha
- Prioritario
- Infusión directa
- Facturación final del año