25.01.2017 1 Grundlagen und Anwendung moderner Trennverfahren Günther K. Bonn Institute of Analytical Chemistry and Radiochemistry, Leopold‐Franzens University of Innsbruck, Innrain 80‐82, A‐6020 Innsbruck, Austria 1 Fällung + ADSI Komplexität von biologischen Proben 1. enorme Komplexität der Proben 2. geringe Konzentration der Zielanalyten 3. beschränkter dynamischer Bereich der analytischen Messgeräte 4. Störkomponenten (Detergenzien, Puffersysteme etc.) MS biologische Probe Herausforderungen: 2 Einleitung Analytik der Biomoleküle
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Grundlagen und Anwendung moderner Trennverfahren · 10 min, 70W microwave‐assisted digest MALDI-MS Proteins Peptides Scheme for Precipitation of Phosphoproteins by Trivalent Europium-,
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25.01.2017
1
Grundlagen und Anwendung moderner Trennverfahren
Günther K. BonnInstitute of Analytical Chemistry and Radiochemistry, Leopold‐Franzens
University of Innsbruck, Innrain 80‐82, A‐6020 Innsbruck, Austria
1
Fällung + ADSI
Komplexität von biologischen Proben
1. enorme Komplexität der Proben
2. geringe Konzentration der Zielanalyten
3. beschränkter dynamischer Bereich der analytischen Messgeräte
according to: Eigel WN et al. 1984 / Fox PF et al. 1998
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Precipitation of Phosphorylated Proteins – Top‐Down Workflow
17
Güzel, Y.; Rainer, M. (); Mirza, M.R.; Bonn, G.K. Highly efficient precipitation of phosphoproteins using trivalent Europium‐,Terbium‐ and Erbium Ions. Analytical and Bioanalytical Chemistry (2012) 403(5), 1323–1331.
Terbium
Wash 1
Wash 2
Pellet
Supernatent
milk
Erbium
Wash 1
Wash 2
Pellet
Supernatent
milk
Europium
PPC‐5 (m/z ~12–13 kDa)
α‐lactalbumin (m/z ~14.1 kDa)
β‐lactoglobulin (m/z ~18.3 kDa)
non‐phosphorylated milk‐proteins
αS1‐casein (m/z ~24.5 kDa)
β‐casein (m/z ~25.1 kDa)
phosphoproteins
αS2‐casein (m/z ~24.5 kDa)
κ‐casein (m/z ~20.0 kDa)
Wash 1
Wash 2
Pellet
Supernatent
milk
Precipitation of Phosphoproteins from Bovine Milk by Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–133118
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Erbium
Wash 1
Wash 2
Pellet
Supernatent
eggwhite
Terbiumegg
white
Pellet
Wash 2
Supernatent
Wash 1
lysozyme (m/z ~14 kDa)
ovomucoid (m/z ~ 28 kDa)
ovoglobulins G2+G3 (m/z ~30‐45 kDa)
ovotransferrin (m/z ~80 kDa)
ovalbumin (m/z ~45 kDa)
non phosphorylated egg‐white proteins phosphoprotein
Europium
Wash 1
Wash 2
Pellet
Supernatent
eggwhite
Precipitation of Phosphoproteins from Egg-White by Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–133119
2M Ln3+‐Chloride
UV/VIS
take supernatant
bicinchoninic acid (BCA)
chelation of two bicinchoninicacid molecules with Cu+1
reduction of Cu+2 to Cu+1 in the presence ofproteins (alkaline conditions)
colorimetric detection 562 nm
Recovery Study of Phosphoprotein
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Methods and instruments
Colorimetric assays: Bradford and BCA
Bradford reagent:Coomassie Brilliant Blue G‐250
BCA assay reaction
Lottspeich F. 2012
Bicinchoninsäure
Absorptionsmaximum von 562 nm
0
20
40
60
80
100
0,5 1 1,5 2 2,5 3
Rec
overy
[%
]
Volume Precipitant [µl]
Lanthanum
Europium
Terbium
Erbium
100%
Precipitation of Phosphoproteins by Trivalent Europium-, Terbium- and Erbium- Ions
Recovery Study
ccasein = 300µg/ml
cprecip. = 2M
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Precipitation of Phosphopeptides by
Trivalent Lanthanide Ions
A Bottum‐Up Approach
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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
Workflow for the precipitation of phosphorylated peptides24
Bottom‐up
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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
MALDI mass spectra taken from digested milk peptides after precipitation with trivalent lanthanide ions. A,phosphopeptide enriched by precipitation with Er3+. B, phosphopeptide enriched by precipitation using Ho3+. C,phosphopeptide enriched by precipitation using Ce3+. α‐S1 and β‐S2 refers to first and second subunits of α‐caseinrespectively. β‐C refers to peptides form β‐casein
Erbium
Holmium
Cer
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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
MALDI mass spectra taken from egg white peptides after precipitation with trivalent lanthanide ions. A,phosphopeptide enriched by precipitation with Er3+. B, phosphopeptide enriched by precipitation using Ho3+. C,phosphopeptide enriched by precipitation using Ce3+. Only phosphorylated peptides are labeled
Erbium
Holmium
Cer
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A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
MALDI mass spectra of a sensitivity study using two synthetic phosphopeptides. A, representing 500 fmol/µL; B, 10 fold dilution (50 fmol/µL) and C, 100 fold dilution (5 fmol/µL)
500 fmol/µL
50 fmol/µL
5 fmol/µL
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High Sensitivity!
[M+H]+ Da Phosphopeptide Sequencesa Phosho‐
groups
ErCl3 HoCl3 CeCl3 LaCl3 EuCl3 TmCl3 TbCl3 TiO2
1254.52
1331.53
1411.50
1466.61
1594.70
1660.79
1832.83
1847.69
1927.69
1951.95
2061.83
2088.89
2432.05
2511.13
2556.10
2619.04
2678.01
2703.50
2720.91
2747.10
2856.50
2901.32
2935.15
2966.16
3008.01
3042.27
3087.99
3122.27
3132.20
EVVGSpAEAGVDAA (Ov‐(340–352))
EQLSpTSpEENSK (α‐S2‐(141–151))
EQLSpTSpEENSK (α‐S2‐(141–151))
TVDMESpTEVFTK (α‐S2‐(153–164))
TVDMESpTEVFTKK (α‐S2‐(153–165))
VPQLEIVPNSpAEER α(‐S1‐(121–134))
YLGEYLIVPNSpAEER (α‐S1)
DIGSESpTEDQAMEDIK (α‐S1‐(58–73))
DIGSESpTEDQAMEDIK (α‐S1‐(58–73))
YKVPQLEIVPNSpAEER (α‐S1‐(119–134))
FQSpEEQQQTEDELQDK (β‐C‐(33–48))
EVVGSpAEAGVDAASVSEEFR (Ov‐(340–359))
IEKFQSpEEQQQTEDELQDK (β‐C‐(33–48))
LPGFGDSpIEAQCGTSVNVHSSLR (Ov‐(62–84))
FQSpEEQQQTEDELQDKIHPF (β‐C‐(48‐67))
NTMEHVSpSpSpEESpIISQETYK (α‐S2‐(17–36))
VNELSpKDIGSpESpTEDQAMEDIK (α‐S1‐(52–73))
LRLKKYKVPQLEIVPNSpAEERL(α‐S1‐(114–135))
QMEAESpISpSpSpEEIVPNSVEAQK (α‐S1‐(74–94))
NTMEHVSpSpSpEESpIISQETYKQ (α‐S2‐(17–37))
EKVNELSpKDIGSpESTEDQAMEDIK (α‐S1‐(50–73))
FDKLPGFGDSpIEAQCGTSVNVHSSLR (Ov‐(59–84))
EKVNELSpKDIGSpESpTEDQAMEDIK (α‐S1‐(50–73))
ELEELNVPGEIVESpLSpSpSpEESITR (β‐C‐(17–40))
NANEEEYSIGSpSpSpEESpAEVATEEVK (α‐S2‐(61–85))
RELEELNVPGEIVESLSpSpSpEESITR (β‐C‐(16–40))
NANEEEYSIGSpSpSpEESpAEVATEEVK (α‐S2‐(61–85))
RELEELNVPGEIVESpLSpSpSpEESITR (β‐C‐(16–40))
KNTMEHVSpSpSpEESpIISQETYKQEK (α‐S2‐(16–39))
Mono
Mono
Di
Mono
Mono
Mono
Mono
Mono
Di
Mono
Mono
Mono
Mono
Mono
Mono
Tetra
Tri
Mono
Penta
Tetra
Di
Mono
Tri
Tetra
Tetra
Tetra
Penta
Tetra
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Recovery of Phosphopeptides
23 20 19 20 21 12 14 18
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PP
P
PP
3.6% HCl
P P
P P
P
trypsin
Tryptic On‐Pellet Digest of Precipitated Phosphoproteins
Inte
nsity
m/z29
Workflow
Development of New Bioanalytical Tools and Methods for the Enrichment of Phosphorylated Peptides and Proteins
Güzel, Y.; Rainer, M. (); Mirza, M.R.; Messner, C.B.; Bonn, G.K. Highly Selective Recovery of Phosphopeptides using Trypsin‐Assisted Digestion of Precipitated Lanthanide‐Phosphoprotein Complexes. Analyst (2013) 138(10), 2897‐2905.
LaPO4
PP
P
PP
washing
LaPO4
Overview of recovered phosphopeptides from a proteinmixture (lysozyme, cytochrome c, myoglobin, bovine serum albumin, a‐ and b‐casein) and bovine milk
proteinmixture milk 1:100 dilution
[M+H]+ Position Protein Phosphopeptide sequences Phospho groups LaCl3 CeCl3 TiO2 LaCl3 CeCl3 TiO2 LaCl3 CeCl31466.6 153‐164 α‐S2 TVDMESpTEVFTK mono + + ‐ + + + + +
Dephosphorylated HeLa cell lysate (1 mg/mL) with spiked α‐ and ß‐casein (5 μg/mL) after enzymatic on‐pellet digestion using trivalent cerium cations. α1, α2 and β correspond to the tryptic phosphopeptides deriving from αS1‐, αS2, and β‐casein, respectively
HeLa cell lysate ‐ digest
spiked cell lysate after precipitation
Development of New Bioanalytical Tools and Methods for the Enrichment of Phosphorylated Peptides and Proteins
On‐pellet digest of precipitated α‐/ß‐casein from spiked cell lysates
crude saliva digest
precipitated fractionCeCl3
Rainer, M. (), Güzel, Y., Messner, C., & Günther Bonn (2014). Co‐Precipitation of Phosphorylated Proteins Using Trivalent Cerium‐, Holmium‐, and Thulium Cations. Current Pharmaceutical Analysis, 10(3), 175‐184.
Development of New Bioanalytical Tools and Methods for the Enrichment of Phosphorylated Peptides and Proteins
On‐pellet digest of precipitated proteins from Human Saliva
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Conclusion
simple and fast method
highly selective for phosphorylated peptides and proteins
enables top‐down and bottum‐up phosphoproteomics
no stationary phase or resin required (reduced unspecific binding)
trypsin was observed to be not affected by the lanthanide ions
the amount of precipitant can be adjusted to each application
MS and LC‐MS compatible
allows automation using liquid handling robotics
Highly efficient precipitation of phosphorylated peptides and proteins using trivalent lanthanide ions
ADSIAnalytische AbteilungVO Moderne Trennmethoden
Innsbruck, 25.01.2017
25.01.2017
18
• Medikamentenentwicklung
• Kosmetik
• Nutrition
Themenschwerpunkte
Phytokosmetik
(Phyto)-NutritionPflanzl.Lebensmittel-
zusatzstoffe
PhytoanalytikPhytopharmazie
Institut für Analytische Chemie
und Radiochemie
allows to see the Nature
Olive‐Net
Themenschwerpunkte „Phytovalley“ Tirol
25.01.2017
19
Medikamentenentwicklung
Ziel: Therapeutisch wirksames Pflanzenextrakt
1. Herstellung und Qualitätskontrolle von Pflanzenextrakten
NEURO‐SYS SAS FranceECOLE NATIONALE D'AGRICULTURE DE MEKNES Morocco
INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE D ALGERIE Algeria
Rangsit University Thailand
ETHNIKO KAI KAPODISTRIAKO PANEPISTIMIO ATHINON Greece
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE France
Natural ingredients and extracts discovery, technical extraction and purification technologies, analytical standards
Biomedical Research
Development of Drug Delivery Systems, API Synthesis, Clinical Studies, Drug Repositioning, Translational Research Novel routes of administration
Drug discovery, neurodegeneration
Development of functional foods and active ingredients from natural substances
Development of novel analytical methods, NMR and evaluation tools for rapid authenticity and quality testing
ICOA; Analytical strategies, molecular interactions and bioactivity tests
Laboratory for green extraction techniques of natural products
Physiology
Analysis, HPLC, CE‐technologysis
Extractions, Analytical methods, HPLC‐MS, MALDI, Screening technologies, Cell systems
Pharmacognosy and Natural Products Chemistry, Plant data base
Agrifood technology
ECOLE NATIONALE D'AGRICULTURE DE MEKNES
Oriental Medicine applications
Medicine and ApplicationsAgriculture
Extraction, Purification, Analysis
Expertise der Projektpartner im Olive‐Net EU‐Projekt
25.01.2017
29
Olive‐Net Roadmap
Olivenöl Abfälle
Olivenkultivierung Olivenölgewinnung
25.01.2017
30
Abfälle
Erzeugung von Wertstoffen aus Olivenöl‐Produktionsabfällen
Tocopherole Aldehyde
Polyphenole Flavonoide
Cyclische Mono‐ und Sesquiterpen‐kohlenwasserstoffe
Wertstoffe für Phytokosmetik‐, Pharma‐, Nahrungsmittel‐, Nahrungsergänzungsmittel
Abfälle
Fester Presskuchen, Waschwasser und einsehr dünnflüssiger Schlamm sind umwelt‐belastende Abfälle bei der Olivenölproduktion
Wertstoffe aus dem Abfall
Mehr als 2 Mio t/a Olivenöl werden in Europa produziert
Im Mittelmeerraum wird die Umwelt dadurchjedes Jahr mit über 30 Millionen TonnenPressrückstände, Waschwasser und Oliven‐blätter belastet.
Dort werden die flüssigen Reststoffe häufig inLagunen und Seen geleitet wodurch dasGrundwasser verunreinigt wird. Die Gewässerverfärben sich schwarz, sterben ab undstinken, da Olivenabfälle auch toxischeVerbindungen enthalten.