2. Gentle ionization mass spectrometry as universal research tool in life science

3. Mass spectrometry: generation and detection of ions Two gentle ionization techniques permit analysis of biomaromolecules MALDIESI Matrix assisted laser desorption ionizationElectrospray ionization López Neyra EEZ

4. MALDIMALDI-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

5. -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

6. 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

7. Use of ESI-MS in life science Syringe pump m/z ratios

8. Not masses but mass/chage ratios are measured Mass spectrum of two peptides Mass spectrum of hemoglobin

9. Micro-heterogenetiy of molecules Krell et al. (1995) Acta Cryst. D53, 612.

10. Heterogeneity: Chicken ovalbumin 59 diferent forms Untreated deglycosylated dephosphorylated deglycosylated and dephosphorylated Yang et al. (2013) Anal. Chem. ;85,12037

11. Analysis of oligonucleotides Reyzer et al. (2001) NAR 29:E103-3. Negative mode

12. 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

13. 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

14. Mass spectrometry & simple Kinetics: stability of phosphorylated phosphoglycerate mutase Question: stability of phosphorylated PGM Krell et al. (1998) J. Peptide Res. 51, 201

15. Mass spectrometry & simple Kinetics: stability of phosphorylated phosphoglycerate mutase The experimental set-up Phosphorylate PGM with 2,3 bisphosphoglycerate Separate phosphorylated PGN from excess 2,3 bisphosphoglycerate Phosphorylated PGM Inject into spectrometer at regular intervals Krell et al. (1998) J. Peptide Res. 51, 201

16. Mass spectrometry & simple kinetics Phosphorylation half-life: 35 minutes T=0 T=18 min Krell et al. (1998) J. Peptide Res. 51, 201

17. Syringe pump Parent Daughter The power of fragmentation

18. The power of fragmentation: glycosylation Glycopeptide Parent ions [M+3H] Daughther ions of 932 Damen et al. (2009) J.Am. Soc. Mass Spec. 20, 2021

19. The power of fragmentation: peptides Peptide Parent ions after fragmentation

20. The power of fragmentation: compounds Paiva-Silva et al. (2006) PNAS 103, 8030 ESI-MS fragmentation spectrum of heme Identification of heme type

21. HPLC – MS/MS HPLC Parent Daughter Separation of compounds (Lourdes) Separation of peptides (peptide mapping)

22. HPLC –Ms/Ms for peptide mapping

23. HPLC –Ms/Ms to identify post-translational modification sites Peptide map of deglycosylated protein Peptide map of glycosylated protein

24. HPLC-MS/MS: The limits Tryptic digest of albumin (67 kDa) Peptides identified by ms are numbered

25. HPLC MS to identify non-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

26. Identification of active site residues 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

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