MALDI Ionisation in der Gasphase: Electron Impact (EI) Chemical Ionisation (CI) Field Ionisation (FI) Ionisation aus Lösungen: Vernebelungs-Techniken Electrospray (ESI) API APCI (Chemical Ionization) APPI (Photoionization) Thermospray API: Atomispheric Pressure Ionisation Ionisationstechniken Desorption-Ionisation Techniken: Desorption mit starkem inhomogenen El. Feld Field-Desorption (FD) Particle Induced Desorption kev - Bombardment: - Secondary Ion Mass Spectrometry (SIMS) - Fast Atom Bombardment (FAB) - Liquid SIMS (LSIMS) MeV-Bombardment - 252 Cf-Plasmadesorption ( 252 Cf-PD) - 127 I-Plasmadesorption ( 127 I-PD) Electron- Bombardment - Electon Avalanche Desorption (EAD) Photon-Bombardment - Laserdesorption (LD) - Matrix-Assisted-Laser-Desorption/ Ionisation (MALDI) 1
Matrix-Assisted-Laser-Desorption- Ionization Quasi-Molekülionen [M + H + ] + [M + Na + ] + [M + K + ] + pos. Ionen-Modus [M + 2H + ] 2+ für große Proteine [M - H + ] - neg. Ionen-Modus Matrix-Assisted-Laser-Desorption- Ionization Quasi-Molekülionen [M + H + ] + [M + Na + ] + [M - H + + 2Na + ] + pos. Ionen-Modus [M - H + ] - [M - 2H + + Na + ] - neg. Ionen-Modus Kohlehydrate neigen z.b. zu Na + und K + Addukten 2
MALDI 4 nsec Puls Plume Vo = 5 m/sec [M + H + ] + Quasi-Molekül-Ionen MALDI-Matrices CO CO HO 2,5-Dihydroxybenzoic acid, DHB CO MeO OMe Sinapinic acid, SA O CN HO α-cyano-4-hydroxycinnamic acid, HCCA 2,4,6-Trihydroxyacetophenone, THAP 3
Wovon hängt es ab, ob eine Matrix für einen bestimmten Analyt (zb bestimmtes Biopolymer) bzw. eine bestimmte Analysenfragestellung geeignet ist? (-) Von der Kristallstruktur und der Größe des Analyts, (-) Ob Analyt in der Matrix stabil ist: d.h. kein on-target Abbau erfolgt (zb durch Hydrolyse säure-labiler Unterstrukturen) (-) Ob Verdampfungs-Event der Matrix zu mehr (heiße Matrix) bzw. minder (kalte Matrix) starkem Auftreten von metastabilen Ionen führt, wodurch die Ausbeute an Fragmentionen beeinflusst wird. MALDI-Matrices HO CO DHB 2,5-Dihydroxybenzoic acid Für: Peptide kleine Proteine Oligonucleotide aber rel. universell heiße Matrix 4
MALDI-Matrices MeO CO OMe Für: Peptide Intakte Proteine (auch große) SA Sinapinic acid MALDI-Matrices CO CN Für: Peptide Glykopeptide HCCA α-cyano-4-hydroxycinnamic acid 5
MALDI-Matrices HO O Für: Peptide Glykopeptide kleine Proteine rel. universell kühlere Matrix THAP 2,4,6-Trihydroxyacetophenone Ionic Liquid Matrix O CH 3 NH H 3 C CH 3 N N HO CH 3 CH 3 Anion: 2,4,6-Trihydroxyacetophonone (THAP) Cation: 1,1,3,3-Tetramethylguanidine 6
Sample.5-1 µl Matrix.5-1 µl Matrix 1 µl Sample 1 µl Dried Droplet Thin Layer MALDI-Analysis of Biopolymers Highly sensitive Well suited for analyte mixtures Reasonable tolerance against salts and some surfactants Fast, easy sample storage, etc., etc. Ionization suppression of certain analytes in mixtures Limited reproducibility in quantitative analysis Limited tolerance against salts and some surfactants Significant extent of undesired analyte fragmentation or degradation Are Ionic Liquid Matrices helpful in these respects? 7
Ionic Liquids: Liquids composed entirely of ions, usually cation and anion being organic ions : Used since about ten years for MALDI analysis of a variety of biopolymers MALDI-Matrices (cryst.) Derivatives of cinnamic acid CO CO Peptides, Glycopeptides CN Proteins α-cyano-4-hydroxycinnamic acid HCCA MeO OMe 4-hydroxy-3,5-dimethoxy cinnamic acid, Sinapinic acid SA CO CO 4-hydroxy-3-methoxycinnamic acid, Ferulic acid FA OMe 4-hydroxycinnamic acid Coumaric acid CumA 8
MALDI-Matrices (cryst.) rel. universal hot matrix CO HO O rel. universal cool matrix 2,5-Dihydroxybenzoic acid DHB HO 2,4,6-Trihydroxy acetophenone THAP ILMs for the analysis of glycostructures in complex mixtures n-butyl-ammonium H 3 N + BuA n-hexyl-ammonium HxA H 3 N + CHCA, DHB, THAP CHCA, DHB, THAP Di-isopropyl-ethylammonium DIEA N,N- tetramethylguanidinium TMG H 3 N + NH H 3 C CH 3 N N 2 + CH 3 CH 3 CHCA, DHB, THAP CumA, DHB, THAP 9
Expected benefits of ILMs Co-crystallization of matrix and analyte not required: Suitability for a wider range of analyte sizes Better homogeneity of the matrix / sample preparation better shot-to-shot reproducibility of intensities Less harsh conditions during Laser-desorption/ionization reduced extent of in-source fragmentation potentially reduced extent of meta-stable ions No acidic conditions on the target Reduced extent of hydrolysis and analyte dissociation Expected benefits of ILMs Co-crystallization of matrix and analyte not required: Suitability for a wider range of analyte sizes Reduced suppression effect of glyco-analytes Better homogeneity of the matrix / sample preparation better shot-to-shot reproducibility of intensities YES NO Less harsh conditions during Laser-desorption/ionization reduced extent of in-source fragmentation YES potentially reduced extent of meta-stable ions NO No acidic conditions on the target YES Reduced extent of hydrolysis and analyte dissociation 1
Types of Ions formed predominantly Solid Crystalline Matrix Positive Ion Mode Negative Ion Mode [M+ H + ] + [M- H + ] - rare Ionic Liquid Matrix [M + H + ] + [M + Na + ] + [M H + + 2Na + ] + [M- H + ] - [M 2H + + Na + ] - Ion Suppression effects (often observed in crystalline matrices) Glycopeptides in peptide mixtures Cleaved glycans in peptide mixtures 11
NH H 3 C CH 3 N N CH 3 CH 3 2 + glycopeptides in peptide mixtures (THAP / TMG-THAP) positive mode AGP tryptic digest 2849.3 THAP pos. mode N 14 38.9 N mc 14 2167.1 C 91 + 5H4N -NH 3 T 128 + 4H3N 2796.1 T 128 + 5H4N 2949.2 TMG-THAP+AP pos. mode %Int. 4 N 53 + 5H4N T 128 + 4H3N Q 13 + 5H4N 2352.6 C 91 + 4H3N -NH -NH 3 Q 3 13 + 4H3N N 53 + 4H3N 243.6 2583.6 N mc -NH 1987.6 14 3 C 91 + 4H3N 2166.8 C 91 + 5H4N -NH 3 C 91 + 5H4N 2812.7 2949.8 T 128 + 5H4N 1 19 2 2 23 24 25 2 27 2 29 3 3 3 Mass/Charge NH H 3 C CH 3 N N CH 3 CH 3 2 + glycopeptides in peptide mixtures (THAP / TMG-THAP) negative mode AGP tryptic digest C 166 mc -NH 3 N 14 N 14 mc 2165.3 C 91 + 5H4N T 128 + 5H4N THAP neg. mode 197.1 37.2 2947.6 1889.1 2811.4 2165.3 TMG-THAP+AP neg. mode %Int. 4 C 166 mc -NH 3 N 14 2368.3 Q 13 + 5H4N / N mc 14 N 53 + 5H4N - NH 3 2165.4 T 2385.3 128 + 5H4N C Q 13 + 5H4N 91 + 5H4N 2351.3 2811.6 2947.7 -NH 3 N 53 + 5H4N C 91 + 5H4N -NH 3 1 19 2 2 23 24 25 2 27 2 29 3 3 3 Mass/Charge 12
Cleaved glycans in peptide mixtures (THAP / TMG-THAP) positive mode AGP tryptic digest after PNGase F treatment 1146.9 1426.1 1214.8 1787.2 THAP pos. mode 1327.9 %Int. W 44 A 62 1146.7 1426. glycan 5H4N 1662.8 TMG-THAP + AP pos. mode 4 V 11 1787.2 N 14 1 1 13 14 15 1 17 1 19 2 2 Mass/Charge Sensitivity and S/N ratio (THAP / TMG-THAP) positive mode AGP tryptic digest 2, shots 6.2 mv 2849.3 THAP pos. mode N 14 38.9 N mc 14 2167.1 C 91 + 5H4N -NH 3 T 128 + 4H3N 2796.1 T 128 + 5H4N 2949.2 %Int. 4 TMG-THAP+AP pos. mode shots 24 mv N 53 + 5H4N T 128 + 4H3N Q 13 + 5H4N 2352.6 C 91 + 4H3N -NH -NH 3 Q 3 13 + 4H3N N 53 + 4H3N 243.6 2583.6 N mc -NH 1987.6 14 3 C 91 + 4H3N 2166.8 C 91 + 5H4N -NH 3 C 91 + 5H4N 2812.7 2949.8 T 128 + 5H4N 1 19 2 2 23 24 25 2 27 2 29 3 3 3 Mass/Charge 13
Extent of fragmentation influenced by matrix Extent of metastable-ion decay is not significantly reduced when using ILMs. Sialic acids from glycopeptides and frequently also NH 3 from peptide ions were cleft off already without CID Extent of in-source/on-target decay is significantly reduced. Example: polymeric saccharides (pullulanes) Pullulans Linear polymers of repeating maltotriose units linked through a 1,6-glycosidic bond 1,6 linkage 1,6 linkage maltotriose unit (three 1,4-linked α-d-glucoses) 14
Detail of Pullulan 5,9 Spectrum H 3 N + DHB 4 497.4 4923.3 5393.5 [M+Na + ] + [M+K + ] + 549.3 4 49 5 5 5 53 54 55 5 57 m/z 1 maltotriose units 11 maltotriose units ILM: BuA-DHB 4 4958.1 [M+Na + ] + 5444.7 [M+ButNH 3 + ] + 4 49 5 5 5 53 54 55 5 57 m/z Pullulan 11,; MALDI-linear TOF DHB extensive in-source (or on-target) decay 1977.9 4 233.7 2629. 8811.6 3279.4 7839.9 9781.6 1749.5 4 m/z ILM: BuA-DHB 4 15 16 7394.8 17 7881.6 18 19 8851.3 8366.7 9336.4 98.5 134.7 number of maltotriose units 21 22 23 1788.3 11269.9 4 m/z 15
Pullulan 22,; MALDI-linear TOF DHB 4 fragments only 5 15 25 m/z ILM: BuA-DHB 4 fragmentation doubly charged 1762.3 15623.9 19.8 18458.5 14667.3 M n = 1595 M w = 14 5 15 25 m/z MALDI Vorteile m/z Bereich bis zu 3. Da Sehr hohe Empfindlichkeit: Niedriger Femtomol Bereich (1-15 ) Weiche D/I, nur geringfügige Fragmentierung Analyse komplexer Gemische ist sehr gut möglich (da kaum Fragmentierung und nur einfach geladene Ionen) Begrenzungen Sehr niedrige Auflösung im m/z-bereich >15. Da Oft störender Matrix-Hintergrund im Spektrum im Bereich unter 7 Da Photoabbau der Analyte durch Laserbestrahlung möglich Diskriminierung schwer ionisierbarer Komponenten häufig Quantifizierung nur bei Einsatz eines Internen Standards möglich 16