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