Grundlagen und Anwendung moderner Trennverfahren · Proteins have a wide variety of amino acids...
Transcript of Grundlagen und Anwendung moderner Trennverfahren · Proteins have a wide variety of amino acids...
Günther K. BonnInstitute of Analytical Chemistry and Radiochemistry, Leopold-Franzens
University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria
Austrian Drug Screening Institute – ADSIInnrain 66, A-6020 Innsbruck, AustriaOffice: Sensengasse, Vienna, Austria
Grundlagen und Anwendung moderner Trennverfahren
Teil 1 - Extraktion
Genome Transcription Proteome Metabolome
Transcription TranslationPost-translational Modification
DNA RNA Proteins Modified Proteins Biological Function
Environment
~30,000 Genes > 1,000,000 ProteinsPathways1500-2000Metabolites
Herausforderung Moderner TrennverfahrenGenomics – Proteomics - Metabolomics
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Genomics
Proteomics
MetabolomicsStationary PhasesMaterial Science
Separation
InstrumentationMS, NMR, Spectroscopy
New Analytical ApproachesBionalysis
DiagnosticsBiomarker Discovery
Purification and Enrichment
Data InterpretationData Analysis
Therapy
Omics - Overview
Stationary PhasesMaterial Science
Separation
Omics - Overview
N Ca
Traditional techniques
(magnetic resonance,Ultrasound, CT…)
SAMPLEbiofluids
Genomic
Proteomic
MetabolomicA
naly
sis NMR, LC-MS,GC-MS, PCR,
Imaging, rt-PCR,ELISA, Chip-Array,…
Screening
DiagnosticsBiomarker Discovery
Therapy
Fällung• Spezifische Fällung von
Phosphoproteinen und Phosphopeptiden
Extraktion
• Festphasenextraktion• Anreicherung von
Proteinen und Peptiden• Entsalzung
Chromato-graphie
• Affinität• Chromatographie• HPLC/UHPLC
Elektro-phorese
• Gel Elektrophorese• Kapillarelektrophorese
Massen-spektro-metrie
• Quadrupol• Ionenfalle• TOF
Moderne Trennverfahren
Überblick
5
What is proteomics
Proteomics is a recent approach to studying proteins
The study of all proteins created or used by an organism
Goal: Identification, quantification and characterisation of all proteins in a given biological systems at a given time point.
6
Proteomics contributes to our understanding of health and disease
Up- and/or down-regulation of protein abundance Protein-protein interactions Post-translational modifications Leakage of proteins from diseased cells or tissues Biomarker discovery
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Bioanalysis - Serum - Biomarker
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Bioanalysis - Complexity of Human Serum
22 proteins are approx. 99% of the whole serum proteome
90% 10%
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Anderson, N. L. (2002) Mol. Cell. Proteomics 1: 845-867
Protein abundance in human plasmaDynamic Range
Warum das Proteom untersuchen ?
Das Genom sagt, was potentiell in einer Zelle passieren könnte,
das Proteom sagt, was tatsächlich passiert.
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Genomics/Proteomics
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Transgenomic Wave-System - Mutation Analysis
Transgenomic Wave HPLC-System
Bonn, Gunther; Huber, Christian; Oefner, Peter. Separation of nucleic acid fragments with alkylated nonporous polymer beads. PCT Int. Appl. (1994), 30 pp.
Mutation Detection by Denaturing HPLC using PackedColumns and Monolithic Capillaries
Packed Column Monolithic Capillary
column: 50 x 4.6 mm I.D. 50 x 0.2 mm I.D.flow rate: 1 ml/min 3.0 µl/mininjection vol.: 5 µl 500 nl
0
157.8 °C
0 5 10time [min]
UV,
254
nmm
AU
UV,
254
nmm
AU
time [min]
0
156 °C
0 2 4 6
Festphasenextraktion - Prinzip
16
Festphasenextraktion - Prinzip
17
Nernst distribution coefficient
Konzentration des Analyten in der stationären Phase
Konzentration des Analyten in der mobilen Phase
Background of chromatographic methods
Important chromatography methods in proteomics
• Ion Exchange Chromatography(IEC)
• Size Exclusion Chromatography(SEC)
• Affinity Chromatography(AC)
• Reversed Phase Chromatography(RPC)
Ion Exchange Chromatography
• Separation is based on charge differences• Reversible interaction between oppositely charged solute and
chromatographic medium• Elution: increasing salt concentration or pH change• Solute molecules are eluted in a concentrated form• Ion exchange types:
– Anion Exchange Chromatography: negatively charged solute molecules compete with negatively charged mobile phase ions for the positively charged sites of the stationary phase
– Cation Exchange Chromatography: positively charged solute molecules compete with positively charged mobile phase ions for the negatively charged sites of the stationary phase.
Ion Exchange Chromatography
Carboxylic acidWeak cation
Sulfonic acidStrong cation
Tertiary amine
Secondary amine
Primary amineWeak anion
Quaternary AmineStrong anion
TypeFunctional groupType of Exchanger
Operating
N+ CH3
NH2
NH
N
SO3-
COO-
Size Exclusion Chromatography
• Solute molecules are separated by their size• Stationary phase has pores of well defined size• Retention is a function of solute penetration into the
pores that is proportional to the hydrodynamic volume of the solute
• No selective interaction with the stationary phase• Particularly useful for buffer exchange
Size Exclusion Chromatography
Affinity Chromatography
• Solute molecules are separated on the basis of specific reversible binding to an affinity ligand attached to the stationary phase.
• Utilizes very specific stationary phases such as antibodies, lectins, etc.
• Desorption is performed by adding a competitive ligand to the elution buffer system, or changing ionic strength, pH or polarity.
• The availability of the affinity ligand defines its applicability.
• Very specific for the solute molecule.
Affinity Chromatography
Most time a spacer is necessary to bind the affinity ligand to the stationary phase
Affinity ligands and applications
LIGAND • Avidin• Aprotinin• Biotin• Concanavalin A• Gelatin• Glutathione• Heparin• Iminoacetic acid• Lysine• Protein A• Phophorylethanolamine• Protein G• Protamine
APPLICATIONS• Biotin derivatives• Serine proteases• Avidin• Glycoproteins, Oligosaccharides• Fibornectine enzymes• Enzymes related to glutathione• Blood coagulation factors• Interferon, serum proteins• Plasminogen, polysaccharides• Human IgG• C-reactive protein• IgG immune complex• IgM
Curtesy of Dr. R. Bishoff
Reversed phase chromatography
• Solute separation is based on reversible hydrophobic interactions with a hydrophobic stationary phase
• Commonly used stationary phases are silica or polymer based with different chain length hydrocarbon ligands
• Due to strong binding, organic solvents are necessary for elution, sometimes with such additives as ion pairing agents.
• During RP-HPLC, proteins may get denatured or loose their biological activity
Je länger ein Stoff in der stationären Phase verbleibt, desto größer wird der
Kapazitätsfaktor und damit auch die Retentionszeit des Analyten. Der Kapazitätsfaktor
gibt an, um wieviel länger sich Moleküle an der stationären Phase im Vergleich zur
mobilen aufhalten. Mit Bruttoretentionszeit (tR) und Totzeit (t0) gilt:
Ein hoher Kapazitätsfaktor beschreibt ein hohes Retentionsverhalten!
Kapazitätsfaktor
Oasis Material (Waters)
Example
Oasis Material (Waters)Alternative to C18
IMACImmobilized metal-ion chromatography
Example
for specific binding of phosphopeptides!
Immobilized metal ion/Metal Chelate affinity chromatography is separation techniquethat is based on coordinate covalent binding between proteins and metal ions. Proteinshave a wide variety of amino acids composition which, in effect, generates a range ofdifferent affinities towards metal ions. However, not many naturally occurring proteinshave affinity for metal ions, so the technique is mainly used to purify recombinantproteins. For example proteins can be engineered to contain a poly-histidine tail(histidine can generally act as a ligand towards divalent metal cations). If the stationaryphase is immobilized with divalent metal cations, a mixture of proteins can beseparated based on their ability to interact with the metal ions. Those proteinscontaining a higher number of histidine residues would be able to bind to the columnmore tightly than those with fewer histidine residues.
Several different types of immobilized metal ion column have been developed toseparate various proteins (e.g. Fe, Co, Cd, Ni, or Zn). Protein separation in IMACgenerally depends on the strength of the metal ion-protein bond. Thus, choosing thetype of immobilized ion is crucial to the success protein separation. By far the mostwidely-used technique is to use an immobilized nickel column, and to engineer poly-histidine tags of six or more residues onto the recombinant proteins of interest. Onething to keep in mind is that the binding between metal ion and protein must bereversible, allowing elution of bounded protein at later steps. Three different elutionstrategies can be applied to IMAC competitive elution, stripping elution and pHAdjustment.
MACHEREY-NAGEL´s concept
Protino Ni-IDA/TEDProtino Ni-IDA/TED – purification of His-tag proteins
Protino Ni-TED
Protino Ni-IDA
TED (tris(carboxymethyl)ethylene diamine)
IDA (iminodiacetic acid)
MOACMetal Oxide Affinity Chromatography
O OP
O OR
TiO2 TiO2
Mechanism: Bridging Bidentate
for specific binding of phosphopeptides!
Example
DHB as “excluder“ or “displacer“
Karl Mechtler et. al
poly(divinylbenzene)
TiO2 < 100 nm
ZrO2 < 100 nm
Preparation of - Hollow MonolithTM
O OP
O OR
TiO2 TiO2Mechanism: Bridging Bidentate
Hollow MonolithTM
Example
Enrichment of Phosphopeptides
... embeddedTiO2/ZrO2
... Phosphorylated Peptides
Enrichment of in vitro phosphorylated ERK1 digest
MALDI MS spectra:
1.) before enrichment (A)
2.) after enrichment with poly(DVB)-TiO2/ZrO2 tips (B)
Signals at m/z 2252.25 and m/z 2332.23 correspond to phosphorylated peptides
Collaboration with Prof. Lukas Huber, Biocenter - Innsbruck
Mono-phosphorylated peptide (m/z 2252.25) identified as RIADPEHDHTGFLTEpYVATRW (SwissProt as database)
MS/MS analysis of enriched phosphopeptides from ERK1 digest
Identification of in vitro phosphorylated ERK1MS/MS Analysis
Activating: ACN/0.1% TFA twice
Equilibrating: 1) H2O/0.1% TFA two times. 2)50% ACN/0.1% TFA containing DHB (20 mg/mL)
Loading: 50% ACN/0.1% TFA containing DHB (20 mg/mL)
Washing: 50% ACN/0.1% TFA containing DHB (20 mg/mL) ,x5Additionally, two washing steps with 80% ACN/0.1% TFA and one washing step with deionized water were performed.
Eluting: 20% ACN/ 50 mM H3PO4, ca 1% NH4OH (pH 10.5)
matrix composition:10 mg/ml DHB80% ACN0.2% H3PO41 % TFA
0
1
2
3
4
5
4x10
Inte
ns. [
a.u.
]
2567.319
1709.807
1502.872
2963.477
2647.3481993.086
2077.040
0
1
2
3
4
5
4x10
Inte
ns. [
a.u.
]
Sample before enrichment
Sample after Ti/Zr-Tipenrichment.8 P-peptides detected
2586.314
ZipTip-MC tips,4 P-peptides detected
2647.1151
2
3
4
5
Inte
ns. [
a.u.
]
1400 1600 1800 2000 2200 2400 2600 2800
m/z
1501.669
1992.682
2587.218
x10 4
0
8 phosphopeptides in p57 protein could be detected after Ti/Zr-Tip enrichment, only 4 with ZipTip MC IMAC
1- 21 2567 GSYPYDVPDYASLEFTVLRPR phosphorylated1- 21 2646 GSYPYDVPDYASLEFTVLRPR 2xphosphorylated
Collaboration with Biocenter Innsbruck
P57 peptides detected after phosphopeptides enrichment#1_2183_ xxxxxx_HAp57wt +src _TiZr
Start - End Observed Mr(expt) Mr(calc) Delta Miss Sequence32 - 44 1485.7451 1484.7378 1484.7260 0.0119 0 R.SLFGPVDHEELSR.E 50 - 60 1272.6811 1271.6738 1271.6106 0.0633 0 R.LAELNAEDQNR.W 50 - 72 2883.3306 2882.3234 2882.2929 0.0304 1 R.LAELNAEDQNRWDYDFQQDMPLR.G Oxidation (M) 50 - 72 2963.3517 2962.3444 2962.2593 0.0852 1 R.LAELNAEDQNRWDYDFQQDMPLR.G Oxidation (M); Phospho (Y) 61 - 72 1709.6695 1708.6622 1708.6593 0.0029 0 R.WDYDFQQDMPLR.G Oxidation (M); Phospho (Y) 61 - 76 2076.8559 2075.8486 2075.8561 -0.0075 1 R.WDYDFQQDMPLRGPGR.L Oxidation (M); Phospho (Y) 77 - 92 1912.9939 1911.9867 1911.9003 0.0864 0 R.LQWTEVDSDSVPAFYR.E 77 - 92 1992.9086 1991.9013 1991.8666 0.0347 0 R.LQWTEVDSDSVPAFYR.E Phospho (Y) 266 - 278 1502.7793 1501.7721 1501.7582 0.0139 1 K.KLSGPLISDFFAK.R Phospho (ST) 287 - 312 2587.1004 2586.0931 2586.1422 -0.0490 0 K.SSGDVPAPCPSPSAAPGVGSVEQTPR.K Phospho (ST)
Automation of Sample Preparation
• Specific enrichment• Purification• Desalting
1. sample loading
laser
2. sample spotting
3. sample analysis 4. data processing
A collaboration with PhyNexus Inc., San Jose, CA, USA
α-caseinPhospho TiZrTiO2/ZrO2PhyNexus
ZipTipMC-Fe3+
Millipore
MonoTipTiO2
GL Sciences
TopTipTiO2
Glygen
TopTipZrO2
Glygen
total number ofphosphopeptides 20 7 11 11 9
ß-caseinPhospho TiZrTiO2/ZrO2PhyNexus
ZipTipMC-Fe3+
Millipore
MonoTipTiO2
GL Sciences
TopTipTiO2
Glygen
TopTipZrO2
Glygen
total number ofphosphopeptides 5 4 5 2 1
Comparative study with commercial products
+
+
[60]fullerenoacetylchloride[811,96]
[60]epoxy fullerene[736,64]
aminopropylSilica fullerene bonded Silica
aminopropylSilica
SiHO
SiOH2N Si
OH
OH
SiHO
SiHO
SiOH2N Si
OH
OH
SiHO
Synthesis of C60-Silica
Fullerene C60-amino silica
fullerene bonded Silica
Example
peptides with multiple phosphorylations – MS
monophosphorylated peptides
Single negatively charged group higher charge states in positive
ionization mode Single loss of phosphoric acid
peptides with multiplephosphorylated amino acids
multiple negatively charged moieties
low charge states preferred multiple losses of phosphoric acid
occur
PO
OHO
O
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
PO
OHO
OP
OOH
O
O
PO
OHO
O
PO
OHO
O
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
Enrichment and Separation of Mono- from Multi-phosphorylated Peptides Utilizing C60-fullerene silica
peptides bind to C60-fullerene silica
1
2
Elution of multi-phosphorylated peptides with 0.1 M NH4OH
3Elution of mono-phosphorylated peptides with 20% ACN and 1% TFA
4
Elution of non-phosphorylated
peptides with 80% ACN and 1 % TFA
Figure 1: Batch experiment, (A) fractionation of multi-phosphorylated peptide by elution with 0.1 M NH4OH and (B) fractionation of mono-phosphorylated peptide by elution 1% TFA in 20% ACN
Enrichment and Separation of Mono- from Multi-phosphorylated Peptides Utilizing C60-fullerene silica
Elution with 0.1 M NH4OH
Elution with 1% TFA in 20% ACN
Elution with 1% TFA in 80% ACN
Enrichment and Separation of Mono- from Multi-phosphorylated Peptides Utilizing C60-fullerene silica
α-casein tryptic digest
Protein Verdau
DTT Dithiothreitol
68
Protein Verdau
(DTT Dithiothreitol)
(CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate)
69
Trypsin Mostly, proteomics scientists use trypsin to
generate peptides− Trypsin cleaves after the positively charged amino
acids arginine [R] and lysine [K], generating multiply positively charged peptides
− Most tryptic peptides have an ideal size for mass spectrometry (500-4000 Da)
− Tryptic peptides fragment very well in the mass spectrometer, generating good quality sequence information
Protein Verdau
70
Peptide Mass Fingerprint
++
++
+++
trypsin
Inte
nsity
m/z
++
++
+++
71
Monolithic Extraction Tips for Enzymatic Digestion
Trypsin in TrisHCl-buffer =
2h , 5°C
GMADVBAIBNToluolDecanol
=+
2h , 95°C
+
BSAα-caseine
+ DTT
Enzyme-Tip
=> =>
+ iodoacetamide
15mins, RT, in dark
15 mins, 60°C
15 mins, 20°C, in
dark
=>
30 mins (in-tip) 60 mins (literature)
DenaturationProtein
Monolithic Extraction Tips for Enzymatic Digestion
Microwave-Assisted In-Tip Digestion
Total Time 45 min!conventional digestion time: 6-10 h
Results - Comparison Study
α-casein digested, eluted from enzyme-Tips with Tip-Technology
Glygen Corp.
Tip
GL SciencesGlygen Corp.
Glygen Corp.
Company
0
10
20
30
40
50
60
70
80
90
a-casein myoglobin BSA
NT1CARTRY
NT1TRY
NT1C18TRY
MonoTip™
GMA/DVB-Tips
sequ
ence
cove
rage
[%]
Desalting of Proteins – Why?
Spin Columns
C18-reversed phase
dialysis
Extraction Tips
Spin Columns
ZipTips (Millipore)
Fullerene C60-Silica for Desalting Biological Samples
Proteines
MALDI-MS spectra of the peptides and proteins which were eluted from Kovasil 300 Å C60-Silica
breakthrough breakthrough
sample sample
elution elution
Peptides
5734.63
8565.632867.10
16952.4014306.6130
1.0
2.0
3.0
4.0
0
1.0
2.0
3.0
4.0
8566.35
14291.4292866.33 16943.00311466.50
0
1.0
2.0
3.0
4.0
1 0
A
B
C5734.73
4282.79
1296
.44
1347
.53
1619
.48
2094
.02
1757
.75
2466
.10
0.0
1.0
2.0
0.0
1.0
2.0
1347
.32
1619
.34
2465
.92
2093
.66
1758
.47
0.0
1.0
2.0
1046.54
1046.72
A
B
C
2000 4000 6000 8000 10000 12000 14000 16000 18000m/z
0.0800 1000 1200 1400 1600 1800 2000 2200 2400
m/z
immobilization washing elution
before
supernatant
elution
before
supernatant
elution
C60-amino-silica
Vallant Rainer M; Szabo Zoltan; Bachmann Stefan; Bakry Rania; Najam-ul-Haq Muhammad; Rainer Matthias; Heigl Nico; Petter Christine; Huck Christian W; Bonn Gunther K. Analytical chemistry (2007), 79(21), 8144-53.
Fullerene C60-amino silica for Desalting
C60-Amino Silica vs. ZipTip® C18 (Millipore)
ZipTips C18supernatant
ZipTips C18elution
1140
.52
1.0
2.0
3.0
4.0
0.0
0.2
0.4
0.6
0.8
5.0
0.0
Inte
nsity
. [a.
u.]
m/z
Phosphopeptide in supernatantNo immobilization on commercial ZipTip ® (Millipore)
Phosphopeptide removed after washingNo immobilization of phosphopeptide
C60-Amino Silicasupernatant
C60-Amino Silicaelution
1.0
2.0
3.0
4.0
5.0
1140
.49
0.0
0.05
0.10
0.15
0.20
0.25
1110 1120 1130 1140 1150 1160 1170 1180
0.0
No Phosphopeptide in supernatantQuantitative binding of phosphopeptideon C60-Amino Silica
Phosphopeptide eluted from C60-Amino Silica
Desalting of hydrophilic Phosphopeptides
Enrichment of Phenolic Compounds using Hexagonal Boron Nitride
Martin Fischnaller, Rania Bakry, Günther Bonn
Institute of Analytical Chemistry and RadiochemistryAustrian Drug Screening Institute – ADSI
University of Innsbruck, Austria
Crystalline forms of Boron Nitride
β or cubic BN
• diamond analog
• stable
• very hard
• black
γ or wurzite BN
• lonsdaleit analog
• only stable under high
pressure
α or hexagonal BN
• graphite analog
• chemically inert
• nontoxic
Boron Nitride, a Novel Material for the Enrichment and Desalting of Protein Digests and the Protein Depletion
N
BN
B
NB
NB
NB
NB
NB
N
B
BN
BN
BN
B
BN
BN
BN
N
BN
0.1446 nm
Covalent bonds
Van der Waalsbonds
0.33
31 n
m
Boron
Nitrogen
Structure of α-BN
Arnold F. Holleman, N. W. Lehrbuch der Anorganischen Chemie; de Gruyter: Berlin, 2007; Vol. 102.
δ 0.93e δ -0.93e
Wettability of α-BN
SEM picture of BN with 60 m² surface area.
Wettability of α-BN
Published in: Hui Li; Xiao Cheng Zeng; ACS Nano 2012, 6, 2401-2409.DOI: 10.1021/nn204661dCopyright © 2012 American Chemical Society
50-67° high wettability
86° low wettability
Snapshots of a water droplet on (A) an atomically BN sheet in the end of classical molecular dynamics
Boron Nitride, a Novel Material for the Enrichment and Desalting of Protein Digests and the Protein Depletion
LC-ESI-MS
Incubation with trypticdigests
MALDI-MS
Washingandenrichment
Elution of peptides
Analysis of desalted and protein free peptide solutions
N
BN
B
NB
NB
NB
NB
NB
N
B
BN
BN
BN
B
BN
BN
BN
N
BN
BN
Boron Nitride, a Novel Material for the Enrichment and Desalting
Bradykinin Substance P Bombesin LHRH Oxytocin
Recovery rates [%]
A B A B A B A B A B
BN 60 71.65 81.67 70.13 73.59 56.12 90.88 92.13 87.46 85.92 95.24
BN 20 98.83 93.74 96.90 90.13 98.71 96.23 95.18 91.01 84.50 92.66
BN NANO 96.75 94.46 108.52 91.30 89.36 95.70 97.07 93.44 91.39 92.85
Graphiteparticle 85.54 83.60 31.70 64.17 47.98 52.78 32.15 76.28 76.33 94.39
Graphite 15 17.62 36.90 14.08 16.78 12.65 17.39 25.70 25.21 49.27 77.04
Recovery Study using different Peptides
A = 60% ACN in 1% TFA and 1% H3PO4; B = 60% ACN in 1% formic acid
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70
BN 60 m²/g
BN 20 m²/g
Graphite 15 m²/g
Cequ [µg/ml]
Mad
s[µ
g/m
g]
Langmuir adsorption isotherms of
Bradykinin for BN 60, BN 20 and
graphite 15
max. loading capacity
mads [µg/mg]
mads/surface area
[µg/m2]
BN 20 m²/g 14.61 0.731
BN 60 m²/g 24.13 0.402
Graphite 15 m²/g 8.19 0.546
Boron Nitride, a Novel Material for the Enrichment and Desalting
MALDI Spectra:
The identified peptides are labeled with
asterisk. Sequence Coverage of desalted
BSA > 68 %
Boron Nitride, a Novel Material for the Enrichment and Desalting
0
20
40
60
80
100
Inte
ns. [
a.u.
]
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Inte
ns. [
a.u.
]
1000 1500 2000 2500 3000 3500m/z
*
*
*
*
*
**
*
*
**
*
*
*
*
*
*
**
*
*
**
* **
*
*
*
*
*
**
*
**
* *
*
*
**
*
*
*
*A
B
x 1000BSA digest in urine
desalted urine sample1000 fold increase in signal intensity
N
BN
B
NB
NB
NB
NB
NB
N
B
BN
BN
BN
B
BN
BN
BN
N
BN
- Pyrrolizidine alkaloids (PA) are secondary plant metabolites (for plant protection)- 400 different PAs in approximately 6000 plant species are known
Contamination of plant products during harvest or through animals (for example bees)Examples for contaminated food: herbal teas, honey, salads etc.
- Problem: Hepatotoxic for animals as well as for humansSafety values for the maximum dose for drugs in Germany:- Application up to 6 weeks: 1 µg/day oral, 100 µg/day cortically - Application for more than 6 weeks: 0.1 µg/day oral, 10 µg/day cortically
Pyrrolizidine alkaloidsBackground
90
- General structure:
- Requirement(s) for toxicity:- 1,2-unsaturated necine- Esterification of at least one OH-group with a branched acid
Nearly all types are in coexistence with their N-oxides
Pyrrolizidine alkaloidsToxicity
91
- HPLC-MS (LOD ~ 1 ppb)- GC-MS (LOD = 3 ppb)
Otonecines and N-oxides (without derivatisation) not detectable- Double antibody ELISA (LOD = 0.1 – 1.5 ppb)
Antibodys only against some specific PAs- HPLC Evaporative Light Scattering Detection (ELSD) (LOD = 40 ppm)- Nonaqueous capillary electrophoresis (NACE) MS (LOD < 7.5 ppm)- Photometric detection with Ehrlich reagent (LOD = 10 ppm)
- General problem for PA analysis: Only about 25 standards are availableValidation is limited to a small spectrum of PAs
Isolation of PAs out of plant material with countercurrent chromatography (CCC) to get morestandards (Cooperation with Medical University of Lublin)
Pyrrolizidine alkaloidsMethods for detection
92
- Detection only possible in the low ppb-range (for GC- and HPLC-MS) for lower concentrations and for separation from other plant substancesenrichment is necessary
- Due to the chemical structure ion exchange is the enrichment method of choice- Automation with PhyNexus MEA 2 possible
PhyTip with Toyopearl SP-650 resin
Pyrrolizidine alkaloidsCation exchange
93
Extraction of the plant material
Enrichment of PAs with ion exchange
Detection with UPLC-MS
Pyrrolizidine alkaloidsProcedure
94
Pyrrolizidine alkaloidsCation exchange – Comparison of commercial products
• In terms of enrichment of pyrrolizidine alkaloids different commercial materials were tested
• Best recoveries (94-99%) were achieved with a polystyrene-divinylbenzene resin functionalized with sulfonate groups
• Further approach- Polystyrene DVB resin in PhyNexus tips and automation of the cation
exchange with PhyNexus MEA 2- Continuing comparison with other resins (in cartridges and tips)- Test of anion exchange resins and Immobilized Metal Ion Affinity
Chromatography (IMAC)
95
Pyrrolizidine alkaloids
• Development of a UPLC-MS/MS method for detection and quantification of standard PA´s
• Improvement of enrichment methodologies with ion-exchange
• Automation of enrichment method
Output
96
SPE of Phenolic Acids using Bismuth Citrate andZirconium Silicate Inside Micro Spin Columns
SPE of Phenolic Acids using Bismuth Citrate andZirconium Silicate Inside Micro Spin Columns
1,3,4,5-tetra-o-galloylquinic acid
CynarinChlorogenic acid
mono-caffeoylquinic-aciddi-caffeoylquinic-acid
Galloyl- and caffeoylquinic-acids are characterized by one free carboxylic group in the quinic acid moiety
Caffeoylquinic acids are the esters of quinic acids with caffeic, ferulic or p-coumaric acids
Galloylquinic acids are the esters of quinic acid with gallic acids
Their biological activity includes anti-HIV, antiasthamatic, bronchial hyper reactivity, allergicreactions and anti-inflamatory properties
Caffeoylquinic acids are antidiabetic, hypoglycemic, antihepatitis, hepatoprotective and anti canceragents
Proposed binding mechanism of bismuth citrate and zirconium silicate with carboxylic group of phenolic acids
Zirconium Silicate45 µm
Bismuth Citrate45 µm
Hussain, Shah, et al. Journal of Pharmaceutical and Biomedical Analysis (2013) in press
Tetragalloyl-quinic acids
Crystalline forms of Boron Nitride
β or cubic BN
• diamond analog
• stable
• very hard
• black
γ or wurzite BN
• lonsdaleit analog
• only stable under high
pressure
α or hexagonal BN
• graphite analog
• chemically inert
• nontoxic
BADGE·2H2O BADGE
BPF BPA
BPZ
Bisphenol derivatives
MW pKa log Kow R2 LOD[ng/ml]
LOQ[ng/ml]
BADGE·2H2O 376.44 14.7b 2.05b 0.999 27.0 82.0
BPF 200.23 7.5a 2.91a 0.999 27.0 82.0BPA 228.29 9.6a 3.32a 0.999 33.0 99.0BPZ 268.35 9.7b 4.53b 0.999 28.0 85.0BADGE 340.41 - 4.02a 0.999 30.0 90.0
Recovery for the enrichment of 5 Phenols
Phenol 4-Nitrophenol 2-Chlorphenol 2-Nitrophenol Dimethylphenol
[12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml] [12.5 µg/ml]
87.51 94.74 108.08 91.29 100.71
93.97 102.95 114.70 95.20 111.82
91.36 101.91 113.24 96.88 105.84
Average 90.95 99.87 112.01 94.46 106.13
RSD 2.66 3.65 2.84 2.34 4.54
5 mg of BN was incubated with a phenol mixture and afterward eluted with80% acetonitrile in water.
• The French Agency for Food, Environmental and Occupational Health & Safety showed that
there are ‘recognized’ effects in animals (effects on reproduction, on the mammary gland, on
metabolism, the brain and behaviour) and other ‘suspected’ effects in humans (on
reproduction, metabolism and cardiovascular diseases). These effects could be observed, even
at low levels of exposure, during sensitive phases of an individual’s development.
• The French Agency for Food, Environmental and Occupational Health & Safety endorses the
conclusions of the Expert Committee on Assessment of the risks related to chemical
substances relating to the risks associated with BPA for human health, and on toxicological
data and data on the use of bisphenols M, S, B, AP, AF, F and BADGE.
Risk associated with bisphenols
Analysis of 5 bisphenol derivatives after enrichment with BN by LC and fluorescence detector
HPLC-FLD chromatogramms of (A) standard solution containing 5 bisphenolderivatives [100 ng/mL] and (B) bisphenol leached from baby bottle afterenrichment with h-BN-60.Chromatographic conditions: Shimadzu LC-10 Acvp, Phenomenex Luna 5u C18 250*4,6 mm, isocratic 3 min 30% B,gradient 30-70% B in 15 min, flow rate 1mL/min, 50°C, inj.: 30 µL,FLD: 230/303 nm
Recovery rates for the enrichment of 5 bisphenolderivatives
Recovery rates ± SD [%]
Concentration[ng/ml] BADGE·2H2O BPF BPA BPZ BADGE
0.5 90.01 ± 5.65 96.43 ± 8.17 111.93 ± 3.8 95.40 ± 5.87 85.38 ± 11.53
1 93.69 ± 2.57 98.88 ± 2.57 98.13 ± 4.05 100.90 ± 1.84 91.43 ± 4.47
2 99.47 ± 1.91 100.92 ± 1.13 95.88 ± 4.21 102.11 ± 3.23 97.48 ± 1.97
10 97.39 ± 2.41 102.47 ± 2.42 103.03 ± 4.21 101.67 ± 2.56 99.17 ± 4.15
100 98.67 ± 5.78 101.55 ± 4.45 101.54 ± 4.47 100.23 ± 2.64 87.51 ± 7.78
Found ± SD [ng/ml]Recovery rates ± SD [%]
BADGE·2H2O BPF BPA BPZ BADGE
river watera nd(92.13 ± 2.69)
nd(102.62 ± 3.73)
nd (107.14 ± 6.50)
nd(104.95 ± 1.92)
nd (98.74 ± 2.57)
drinking watera nd(90.16 ± 2.40)
nd(98.56 ± 3.51)
nd (99.16 ± 1.60)
nd (101.14 ± 2.58)
nd(96.68 ± 3.35)
colab 4.63 ± 0.14 (103.41 ± 4.39)
2.23 ± 0.21 (96.98 ± 2.39)
10.34 ± 0.41(96.93 ± 5.72)
nd (92.46 ± 6.58)
1.49 ± 0.04 (96.57 ± 3.84)
apple juiceb 1.93 ± 0.15(96.43 ± 3.63)
3.11 ± 0.30(90.77 ± 3.63)
nd (84.22 ± 2.20)
nd (98.74 ± 1.23)
1.36 ± 0.22(98.22 ± 2.71)
lemon sodab 8.14 ± 0.09(82.46 ± 3.01)
1.40 ±0.07(84.45 ± 5.44)
nd (97.60 ± 1.81)
nd (87.61 ± 2.94)
5.70 ± 0.04(94.00 ± 2.61)
citrus soft drinkb 5.32 ± 0.71 (103.90 ± 2.58)
nd(97.82 ±5.82)
1.04 ± 0.31(98.31 ± 5.67)
nd (98.05 ± 4.04)
1.56 ± 0.042 (99.63 ± 4.42)
herbal sodab nd (100.45 ± 5.15)
nd(99.07 ± 5.09)
nd (104.43 ± 6.20)
nd (105.07 ± 7.21)
nd (108.42 ± 4.83)
energy drinkb 4.46 ± 0.07 (98.54 ± 9.21)
1.87(91.55 ± 4.21)
5.57 ± 0.81(93.55 ± 5.84)
nd (100.76 ± 2.04)
nd (103.60 ± 1.91)
canned mushroom liquidb 20.42 ± 1.19(99.06 ± 6.88)
nd(91.35 ±0.79)
8.60 ± 0.37 (98.47 ± 8.37)
nd (103.71 ± 6.17)
1.81 ± 0.12 (99.63 ± 0.62)
pickled cucumber liquidb 1.81 ± 0.15 (97.53 ± 1.12)
2.23 ± 0.21(96.02 ± 2.45)
125.00 ± 2.18(109.12 ± 0.77)
nd (103.36 ±3.80)
1.44 ± 0.06 (92.67 ± 0.71)
pickled onion liquidb 2.98 ± 0.08 (105.80 ± 3.90)
nd(96.53 ± 8.20)
13.45 ± 0.63 (92.03 ± 4.70)
nd(97.91 ± 3.12)
1.74 ± 0.00 (95.20 ± 0.63)
urineb nd (100.45 ± 3.13)
nd(105.67 ± 0.68)
nd(92.80 ± 2.52)
nd(105.26 ± 4.00)
nd(103.70 ±3.45)
Determination of bisphenol derivatives
Leaching of BPA from polycarbonate products
leached BPA [ng/ml] ± SD (%)
Babybottle 1. treatment 20.79 1.05
Babybottle 2. treatment 19.82 0.72
muffin form 2.26 0.07
Siringe 0.85 0.03
Treatment conditions: boiling water for 1 h
Langmuir adsorption isotherms of Bisphenol A on h-BN-60
M ad
s[µ
g/m
g]
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30
Cequ[µg/ml]
max. loading capacity of Bisphenol A = 60 µg/mg
Depletion of proteins with BN
0
1000
2000
3000
4000
5000
Inte
ns. [
a.u.
]
*
0
1
2
3
4
5
4x10
Inte
ns. [
a.u.
]
1000 1500 2000 2500 3000 3500m/z
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2000
4000
Inte
ns. [
a.u.
]
30000 40000 50000 60000 70000m/z
2000
4000Inte
ns. [
a.u.
]
30000 40000 50000 60000 70000m/z
A
B
x 10
Seru
m a
lbum
inIs
ofor
m 1
[M+2
H] 2+
Seru
m a
lbum
inIs
ofor
m 2
(A) MALDI-MS analysis of a human albumin proteinand those digest in a ratio of 50000-1 (1mg/ml-0,02 µg/ml) and a image of thecrystallization of the sample and matrixSequence coverage of HSA 36%
(B) MALDI spectra of the sample after SPE with BN(sequence coverage of 72%) and an image ofthe matrix crystallization
asterisk: Mascot identified peptides
Aufgabe 1
Analyt: Disaccharide
Welche Festphase? Warum?
Aufgabe 2
Analyt: Proteine
Welche Festphase? Warum?
Aufgabe 3
Analyt: Pestizide
Welche Festphase? Warum?