ESI and MALDI Mass Spectrometry. S. Sankararaman Department of Chemistry Indian Institute of Technology Madras Chennai

Transcription

1 ESI and MALDI Mass Spectrometry S. Sankararaman Department of Chemistry Indian Institute of Technology Madras Chennai

2 MODULE 22 ESI-Q MASS SPECTROMETRY - examples

3 Mol. Wt = 1140 ESI-MS of antibiotic gramicidine formyl-l-x-gly-l-ala-d-leu-l-ala-d-val- L-Val-D-Val-L-Trp-D-Leu-L-Y-D-Leu-L-Trp- D-Leu-L-Trp-ethanolamine X = valine or isoleucine and Y = tryptophan [M+H + ] (m/z 1141, z=1) and [M+2H + ] (m/z 571, z=2) are seen

4 ESI-MS of polyethylene glycol (PEG) The nomial molecular weight correspond to the most abundant oligomer of PEG I and II correspond to mono and doubly charged ions

5 ESI-MS of polyethylene glycol (PEG) of various molecular weight I, II, III, IV etc correspond to the charge on the molecular ion The nomial molecular weight correspond to the most abundant oligomer of PEG

6 ESI- mass spectra of protein samoles Protein samples in acidified water, ethanol and isopropanol mixture. The adducts are protons

7 ESI- mass spectra of protein samoles Protein samples in acidified water, ethanol and isopropanol mixture. The adducts are protons

8 ESI-MS of a synthetic oligonucleotide negative ion mode Mol. Wt 4260

9 [M+100H + ] Z = 100 Observed m/z = 1331 ESI-MS of bovine serum albumin dimer. MW = 133,000

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11 Positive ESI-MS m/z spectrum of the protein hen egg white lysozyme. Next slide for calculating M (mass) and n (the charge on the ion)

12 Calculating the molecular weight from ESI-MS If the ions appearing at m/z in the lysozyme spectrum have "n" charges, then the ions at m/z will have "n+1" charges, (due to protonation) = (MW + nh+)/n and = [MW + (n+1)h+] /(n+1) These simultaneous equations can be rearranged to exclude the MW term: n(1431.6) - nh+ = (n+1) (n+1)h+ n(1431.6) = n(1301.4) H+ n( ) = H+ and so: n = ( H+) / ( ) hence the number of charges on the ions at m/z = /130.2 = 10. Putting the value of n back into the equation: = (MW + nh+) n gives x 10 = MW + (10 x 1.008) and so MW = 14, therefore MW = 14,305.9 Da

13 Molecular mass profile of lysozyme obtained by maximum entropy processing of the m/z spectrum

14 Proteins in their native state, or at least containing a significant amount of folding, tend to produce multiply charged ions covering a smaller range of charge states (say two or three). These charge states tend to have fewer charges than an unfolded protein would have, due to the inaccessibility of many of the protonation sites. In such cases, increasing the sampling cone voltage may provide sufficient energy for the protein to begin to unfold and create a wider charge state distribution centering on more highly charged ions in the lower m/z region of the spectrum. ESI-MS of native state protein and unfolded protein are different!!

15 Positive ESI-MS m/z spectra of the protein apo-pseudoazurin analysed in water at ph7 (upper trace) and in 1:1 acetonitrile:0.1% aq. formic acid at ph2 (lower trace).

16 (refer to spectra in the previous slide) Analysis of the protein in 1:1 acetonitrile : 0.1% aqueous formic acid at ph2 gave a Gaussian-type distribution with multiply charged states ranging from n = 9 at m/z to n = 19 at m/z 705.3, centering on n = 15 (lower trace). The molecular mass for this protein was 13,381 Da. Analysis of the protein in water gave fewer charge states, from n = 7 at m/z to n = 11 at m/z , centering at n = 9 (upper trace). Not only has the charge state distribution changed, the molecular weight is now 13,444 Da which represents an increase of 63 Da and indicates that copper is remaining bound to the protein. Many types of protein complexes can be observed in this way, including protein-ligand, protein-peptide, protein-metal and protein-rna macromolecules.

17 From our lab: identification of macrocycles as Ag + complex MeO OMe n Macrocycle Yield (%) MeO OMe n 1 Dimer 16 2 Trimer 10 3 Tetramer 33 4 Pentamer 8 5 Hexamer 13 6 Heptamer Octamer 6 S. Sankararaman, A. Bandyopadhyay J. Org. Chem. 2006, 71,

18 MeO OMe MeO MeO OMe OMe MeO OMe MeO OMe NMR is not useful for characterization due to similar spectra for all sizes of the macrocycle

19 ESI-MS OF Ag + COMPLEXES OF HEXAMER calculated experimental The cluster of peaks is due to isotopes of Ag+ in the [M+Ag + ] adduct

20 ESI-MS OF Ag + COMPLEXES OF OCTAMER calculated experimental

21 n Macro cycle HRMS calc.for HRMS found 2 Trimer C 36 H 30 O 6 Ag Tetramer C 48 H 40 O 8 Ag Pentamer C 60 H 50 O 10 Ag Hexamer C 72 H 60 O 12 Ag Heptamer C 84 H 70 O 14 Ag Octamer C 96 H 80 O 16 Ag

22 THANK YOU