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Desalting of Proteins – Why?
Spin Columns
C18-reversed phase
dialysis
Extraction Tips
Spin Columns
ZipTips (Millipore)
N
H
OH
SiHO
SiHO
SiOSi
OH
OH
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
SPESPESPEPart 2 Part 2
5734.63
8565.63
2867.1016952.4014306.613
0
1.0
2.0
3.0
4.0
0
1.0
2.0
3.0
4.0
8566.35
14291.429
2866.33 16943.00311466.50
0
1.0
2.0
3.0
4.0
8566.83
2866.56
4282.5911466.222 14291.426
0.0
0.2
0.4
0.6
0.8
1.0
2000 4000 6000 8000 10000 12000 14000 16000 18000
A
B
C
D
5734.73
5734.43
4282.79
m/z
4x
10
Inte
ns
ity
. [a
.u.]
129
6.4
4
134
7.5
3
161
9.4
8
209
4.0
2
175
7.7
5
246
6.1
0
0.0
1.0
2.0
0.0
1.0
2.0
134
7.3
2
161
9.3
4
246
5.9
2
209
3.6
6
175
8.4
7
0.0
1.0
2.0
161
9.4
5
134
7.4
2
246
6.0
6
175
8.5
5
209
3.7
8
0.0
1.0
2.0
800 1000 1200 1400 1600 1800 2000 2200 2400
1046.54
1046.72
1047.39
m/z
A
B
C
D
Peptide Proteine
SPESPESPEPart 2 Part 2
5734.63
8565.63
2867.1016952.4014306.613
0
1.0
2.0
3.0
4.0
0
1.0
2.0
3.0
4.0
8566.35
14291.429
2866.33 16943.00311466.50
0
1.0
2.0
3.0
4.0
8566.83
2866.56
4282.5911466.222 14291.426
0.0
0.2
0.4
0.6
0.8
1.0
2000 4000 6000 8000 10000 12000 14000 16000 18000
A
B
C
D
5734.73
5734.43
4282.79
m/z
4x
10
Inte
ns
ity
. [a
.u.]
129
6.4
4
134
7.5
3
161
9.4
8
209
4.0
2
175
7.7
5
246
6.1
0
0.0
1.0
2.0
0.0
1.0
2.0
134
7.3
2
161
9.3
4
246
5.9
2
209
3.6
6
175
8.4
7
0.0
1.0
2.0161
9.4
5
134
7.4
2
246
6.0
6
175
8.5
5
209
3.7
8
0.0
1.0
2.0
800 1000 1200 1400 1600 1800 2000 2200 2400
1046.54
1046.72
1047.39
m/z
A
B
C
D
Peptide Proteine
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 C18
supernatant
ZipTips C18
elution
11
40
.52
1.0
2.0
3.0
4.0
0.0
0.2
0.4
0.6
0.8
5.0
0.0
Inte
nsi
ty. [
a.u
.]
m/z
Phosphopeptide in supernatant
No immobilization on commercial
ZipTip ® (Millipore)
Phosphopeptide removed after washing
No immobilization of phosphopeptide
C60-Amino Silica
supernatant
C60-Amino Silica
elution
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 supernatant
Quantitative binding of phosphopeptide
on 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 Radiochemistry
Austrian 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
0.1446 nm
Covalent bonds
Van der Waalsbonds
0.3
33
1 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/nn204661d
Copyright © 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 tryptic
digests
MALDI-MS
Washing
and
enrichment
Elution of
peptides
Analysis of desalted
and protein free
peptide solutions
N
B
N
B
N
B
N
B
N
B
N
B
N
B
N
B
B
N
B
N
B
N
B
B
N
B
N
B
N
N
B
N
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
Graphite particle
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]
Mads
[µg/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 1000
BSA digest in urine
desalted urine sample 1000 fold increase in signal intensity
N
B
N
B
N
B
N
B
N
B
N
B
N
B
N
B
B
N
B
N
B
N
B
B
N
B
N
B
N
N
B
N
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
*
***
***
**
* **
*
**
*
*
**
**
**
**
***
**
**
**
**
**
** *
*
** *
**
**
*
*
**
**
2000
4000
Inte
ns.
[a.
u.]
30000 40000 50000 60000 70000m/z
2000
4000
Inte
ns.
[a.
u.]
30000 40000 50000 60000 70000
m/z
A
B
x 10
Ser
um
alb
um
in
Isofo
rm 1
[M+
2H
] 2+
Ser
um
alb
um
in
Isofo
rm 2
(A) MALDI-MS analysis of a human albumin protein and those digest in a ratio of 50000-1 (1 mg/ml-0,02 µg/ml) and a image of the crystallization of the sample and matrix
Sequence coverage of HSA 36%
(B) MALDI spectra of the sample after SPE with BN (sequence coverage of 72%) and an image of the matrix crystallization
asterisk: Mascot identified peptides
15
‐ 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 humans Safety 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 alkaloids Background
16
‐ 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 alkaloids Toxicity
17
‐ 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 available Validation is limited to a small spectrum of PAs
Isolation of PAs out of plant material with countercurrent chromatography (CCC) to get more standards (Cooperation with Medical University of Lublin)
Pyrrolizidine alkaloids Methods for detection
18
‐ Detection only possible in the low ppb-range (for GC- and HPLC-MS) for lower concentrations and for separation from other plant substances enrichment 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 alkaloids Cation exchange
19
Extraction of the plant material
Enrichment of PAs with ion exchange
Detection with UPLC-MS
Pyrrolizidine alkaloids Procedure
20
Pyrrolizidine alkaloids Cation 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)
21
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
SPE of Phenolic Acids using Bismuth Citrate and Zirconium Silicate Inside Micro Spin Columns
SPE of Phenolic Acids using Bismuth Citrate and Zirconium Silicate Inside Micro Spin Columns
1,3,4,5-tetra-o-galloylquinic acid
Cynarin Chlorogenic acid
mono-caffeoylquinic-acid di-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, allergic
reactions and anti-inflamatory properties
Caffeoylquinic acids are antidiabetic, hypoglycemic,
antihepatitis, hepatoprotective and anti cancer agents
Proposed binding mechanism of bismuth citrate and zirconium silicate with carboxylic group of phenolic acids
Zirconium Silicate
45 µm
Bismuth Citrate
45 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
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 with
80% acetonitrile in water.
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 82.0
BPA 228.29 9.6a 3.32a 0.999 33.0 99.0
BPZ 268.35 9.7b 4.53b 0.999 28.0 85.0
BADGE 340.41 - 4.02a 0.999 30.0 90.0
• 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.
• Endokrine Disruptoren
Risk associated with bisphenols
Tolerable Daily Intake (TDI) of 50 µg BPA/kg body weight (b.w.)/day as set by EFSA in 2006.
The migration limit of BPA set by European Union of 600 µg/kg food.
Tolerable Daily Intake (TDI) of 150 µg BADGE and its hydrolytic products/kg body weight (b.w.)/day.
The migration limit of BADGE and its hydrolytic products 9000 µg/kg food.
http://www.efsa.europa.eu/en/topics/topic/bisphenol.htm
http://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htmhttp://www.efsa.europa.eu/en/topics/topic/bisphenol.htm
Analysis of 5 bisphenol derivatives after enrichment
with BN by LC and fluorescence detector
HPLC-FLD chromatogramms of (A) standard solution containing 5 bisphenol
derivatives [100 ng/mL] and (B) bisphenol leached from baby bottle after
enrichment 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 bisphenol
derivatives
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
Glas
Dose
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
Syringe 0.85 0.03
Treatment conditions: boiling water for 1 h
Langmuir adsorption isotherms of Bisphenol A
on h-BN-60
M a
ds
[µg/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
Aufgabe 1
Analyt: Disaccharide
Welche Festphase? Warum?
Aufgabe 2
Analyt: Proteine
Welche Festphase? Warum?
Aufgabe 3
Analyt: Pestizide
Welche Festphase? Warum?
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
40
Teil 3 - Fällung
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:
41
Einleitung Analytik der Biomoleküle
Matthias Rainer
42
22 Proteine machen 99% des gesamten Serumproteoms aus!
90% 10%
Einleitung Analytik der Biomoleküle
1. Enorme Komplexität von biologischen Proben
z.B. Blutserum
Matthias Rainer
43 Anderson, N. L. (2002) Mol. Cell. Proteomics 1: 845-867
Dynamischer Bereich von Blutplasma
Einleitung Analytik der Biomoleküle
2. Geringe Konzentration vieler Zielanalyten
Selektivität
Empfind-lichkeit
Geschwin-digkeit
Hoher Probendurchsatz
Verbesserte Detektion
Gezielte Analyse
Effizienz
Effiziente Probenvorbereitung
Einleitung Analytik der Biomoleküle
Matthias Rainer
Zusätzliche Komplexität durch Variationen von Proteinen
• Bei einem Pentapeptid (Länge 5 Aminosäuren) gibt es 205 = 3.200.000
Kombinationsmöglichkeiten
• Proteine haben eine Länge von 100 bis 1000 Aminosäuren, also 20100 -
201000 Kombinationsmöglichkeiten
• Drehbarkeit der C-C Bindung in der Kette
• Peptidbindung normalerweise in trans-, kann aber auch in cis-
Konformationen vorliegen
• Seitengruppen können modifiziert sein
• Posttranslationale Modifikationen (z.B. Glykosylierung, Phosphorylierung)
45
Einleitung Analytik der Biomoleküle
Das Verhalten von Proteinen in wässriger Lösung hängt von
der Art des Lösungsmittels ab, denn in Gegenwart von Salzen,
Säuren oder Laugen ändert sich das Löslichkeitsverhalten der
Proteine stark. Viele Methoden zur Trennung von Proteinen
machen sich diese Eigenschaft zunutze, so z.B. bei der Fällung
von Proteinen aus einer Lösung.
Fällung von Proteinen
46
Proteine können auf verschiedene Art in ihrer nativen Form gefällt werden:
• Fällung durch Aussalzen
• isoelektrische Fällung (Fällung am IEP)
• Fällung mit organischen Lösungsmitteln
• Co-Präzipitation
Fällung von Proteinen
47
Proteine können auf verschiedene Art in ihrer nativen Form gefällt werden:
• Fällung durch Aussalzen
Fällung von Proteinen
Das Aussalzen von Proteinen ist ein Spezialfall einer Fällungsreaktion, die als Trennverfahren bei der Reinigung von Proteinen eingesetzt wird. Dabei nutzt man aus, dass Proteine nur dann in Lösung vorliegen, wenn sie über eine ausreichende Hydrathülle aus Wassermolekülen verfügen. Werden der Lösung Salze zugesetzt, so binden die dissoziierten Ionen ihrerseits Wassermoleküle in ihrer Hydrathülle und entziehen diese den Proteinmolekülen. Ab einer bestimmten Salzkonzentration, die von der Art des Salzes und dem Protein abhängt, wird dieser Effekt so stark, dass das Protein nicht mehr in Lösung gehalten werden kann, d.h. es fällt aus der Lösung aus und bildet einen Niederschlag.
48
Proteine können auf verschiedene Art in ihrer nativen Form gefällt werden:
• isoelektrische Fällung (Fällung am IEP)
Fällung von Proteinen
Praktisch lässt sich der IEP auch zur Fällung von Proteinen aus einer Lösung nutzen. Die Löslichkeit eines Proteins wird sehr stark durch den pH-Wert der Umgebung beeinflusst und erreicht am isoelektischen Punkt ein Minimum. Ober- oder unterhalb des IEP tragen alle Moleküle die gleiche Ladung (positiv oder negativ) und stoßen sich daher ab. Eine Zusammenballung zu unlöslichen Aggregaten ist durch die Abstoßung der Moleküle untereinander verhindert und das Protein bleibt in Lösung.
49
Proteine können auf verschiedene Art in ihrer nativen Form gefällt werden:
• Fällung mit organischen Lösungsmitteln
Fällung von Proteinen
Aceton Precipitation
TCA Precipitation
Wessel Flügge Precipitation
50
51
Fällung von Proteinen
Fällung mit organischen Lösungsmitteln
52
Fällung von Proteinen
Fällung mit organischen Lösungsmitteln
53
Fällung von Proteinen
Fällung mit organischen Lösungsmitteln
Proteine können auf verschiedene Art in ihrer nativen Form gefällt werden:
• Co-Präzipitation
Fällung von Proteinen
54
55
56
57
58
59
60
Precipitation of Phosphoproteins by
Trivalent Lanthanide Ions
A Top-Down Approach
61
Protein phosphorylation
Protein dephosphorylation
Berg JM et al. 2010
Protein separation and isolation
2. Chromatographic techniques
IMAC Immobilized Metal ion Affinity Chromatography
MOAC Metal Oxide Affinity Chromatography
Berg JM et al. 2010 According to: Leitner A. 2010
Proposed Precipitation Mechanism
64
Lanthanide Phoshates (Solubility Products)
solubility products
[logKsp(M)]
ErPO4 -25.13 ± 0.11
EuPO4 -25.96 ± 0.03
TbPO4 -25.39 ± 0.04
Solubility products of the rare-earth phosphates
References:
Xuewu Liu and Robert H. Bryne, Geochimica et Cosmichimica Acta, 1997, Vol. 61,No8,pp. 1625-1633
Li, X. et al. Geochimica et cosmochimica acta,1997.61(8):p.1625-1633
65
Dissolvation
of pellet
MALDI-MS
on-pellet
digest
denaturation, tryptic digestion
30% formic acid
washing
precipitation
10 min, 70W microwave-assisted digest
MALDI-MS
Peptides Proteins
Scheme for Precipitation of Phosphoproteins by
Trivalent Europium-, Terbium- and Erbium- Ions
Precipitant
KH2PO4
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–1331 66
Methods and instruments
MALDI-TOF MS: linear mode
Methods and instruments
MALDI-TOF MS: reflector mode
sign
al in
ten
sity
[a.
u.]
m/z
Erbium Protein
Standard
Wash 1
Wash 2
Pellet
Supernatent
sig
na
l in
ten
sity
[a
.u.]
m/z
Terbium
Wash 1
Wash 2
Pellet
Supernatent
Protein
Standard
αS1-casein (m/z ~24.5 kDa)
β-casein (m/z ~25.1 kDa)
Phosphoproteins cytochrom c (m/z ~11.7 kDa)
lysozyme (m/z ~14.4 kDa) myoglobin (m/z ~17 kDa)
BSA (m/z ~ 69 kDa)
Standardproteins
sig
na
l in
ten
sity
[a
.u.]
m/z
Europium
Wash 1
Wash 2
Pellet
Supernatent
Protein
Standard
Precipitation of Phosphoproteins from Standards by
Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–1331 69
Standart Eu
0
1
2
3
4
5
4x1
0In
tens.
[a.u
.]
1000 2000 3000 4000 5000 6000 Dam/z
β
β β
β
β
α1
β
ββ
α1
α1
α1
α1
α1
α1
α1
α1
α1
α1
α1
α-casein seq. Coverage
[%] Mascot Search
Score Error [ppm]
Er 94 90 117
Eu 94 79 106
Tb 92 98 117
β-casein seq. Coverage
[%] Mascot Search
Score Error [ppm]
Er 56 76 32
Eu 67 54 94
Tb 53 75 147
Europium
Peptide Mass-Fingerprint-Analyse
α1... αS1-Casein
β … β-Casein
On-Pellet Digest of Precipitated Phosphoproteins by
Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–1331 70
Bovine milk composition
87,1%
4,9% 3,9%
3,4% 0,7%
water
carbohydrates
fats
proteins
minerals
38%
9% 29%
9%
4% 9% 2%
α-S1-casein
α-S2-casein
β-casein
κ-casein
α-lactalbumin
β-lactoglobulin
others
according to Pavia DL. 1990 / Eigel WN et al. 1984
Milk proteins
protein (variant) mol. weight /
Da amino acids
c / g∙L-1 P
per mol SH | S-S per mol
pI
αS1-casein (B 8P) 23,614 199 12 – 15 8 0 | 0 4.4 – 4.8
αS2-casein (A 11P) 25,230 207 3 – 4 11 2 | 0 −
β-casein (A2 5P) 23,983 209 9 – 11 5 0 | 0 4.8 – 5.1
κ-casein (B 1P) 19,023 169 2 – 4 1 2 | 0 5.3 – 5.8
α-lactalbumin (B) 14,176 123 0.6 – 1.7 0 0 | 4 4.2 – 4.5
β-lactoglobulin (B) 18,363 162 2 – 4 0 1 | 2 5.1
serum albumin 66,267 582 0.4 0 1 | 17 4.7 – 4.9
according to: Eigel WN et al. 1984 / Fox PF et al. 1998
sig
na
l in
ten
sity
[a
.u.]
m/z
Terbium
Wash 1
Wash 2
Pellet
Supernatent
milk
sig
na
l in
ten
sity
[a
.u.]
m/z
Erbium
Wash 1
Wash 2
Pellet
Supernatent
milk
sig
na
l in
ten
sity
[a
.u.]
m/z
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) 403:1323–1331 73
0.0
0.5
1.0
1.5
2.0
2.5
4x1
0In
tens.
[a.u
.]
1000 2000 3000 4000 5000 6000m/z
α1
κ β
κ
Da
κ
α1
α1α1
α1
α2
α1
α1
α1
ββ β
ββ
α2
α2
α2
α2
α2
α2
β
Milk Tb
Eu seq. Coverage [%] Mascot Search Score Error [ppm]
α(S1)-casein 95 74 81 α(S2)-casein 76 68 55
β-casein 25 72 53 κ-casein 50 68 46
Tb seq. Coverage [%] Mascot Search Score Error [ppm]
α(S1)-casein 95 69 98 α(S2)-casein 51 74 96
β-casein 46 69 77 κ-casein 50 56 118
Er seq. Coverage [%] Mascot Search Score Error [ppm]
α(S1)-casein 98 70 74 α(S2)-casein 51 56 56
β-casein 25 55 44 κ-casein 50 60 68
Terbium
α1... αS1-Casein
α2... αS2-Casein
β … β-Casein
κ … κ-Casein
Peptide Mass-Fingerprint-Analysis
On-Pellet Digest of precipitated Phosphoproteins from Bovine Milk
by Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–1331 74
sig
na
l in
ten
sity
[a
.u.]
m/z
Erbium
Wash 1
Wash 2
Pellet
Supernatent
egg
white
sig
na
l in
ten
sity
[a
.u.]
m/z
Terbium egg
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
sig
na
l in
ten
sity
[a
.u.]
m/z
Europium
Wash 1
Wash 2
Pellet
Supernatent
egg
white
Precipitation of Phosphoproteins from Egg-White by
Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–1331 75
ov
ov
ov
ov
ov
ov
ot
ov
ov
ot
otov
ot
ov
0.0
0.2
0.4
0.6
0.8
1.0
x10
Inte
ns. [
a.u.
]
1000 1500 2000 2500 3000 3500 4000 4500 5000 5500
m/zDa
1.2
ot
ov
ov
ot
ov
ov ov
ot
ot
ov
Eu seq. Coverage [%] Mascot search
Score Error [ppm]
ovalbumin 74 62 169
ovotransferrin 52 118 153
Tb seq. Coverage [%] Mascot search
Score Error [ppm]
ovalbumin 59 75 169
ovotransferrin 58 113 175
Er seq. Coverage [%] Mascot search
Score Error [ppm]
ovalbumin 60 113 172
ovotransferrin 80 66 184
Europium Peptide Mass-Fingerprint-Analysis
ov... Ovalbumin
ot... Ovotransferrin
On-Pellet Digest of precipitated Phosphoproteins from Egg-
White by Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–1331 76
2M Ln3+-Chloride
UV/VIS
take supernatant
bicinchoninic acid (BCA)
chelation of two bicinchoninic acid molecules with Cu+1
reduction of Cu+2 to Cu+1 in the presence of proteins (alkaline conditions)
colorimetric detection 562 nm
Recovery Study of Phosphoprotein
77
Methods and instruments
Colorimetric assays: Bradford and BCA
Bradford reagent: Coomassie Brilliant Blue G-250
BCA assay reaction
Lottspeich F. 2012
0
20
40
60
80
100
0,5 1 1,5 2 2,5 3
Re
co
ve
ry [
%]
Volume Precipitant [µl]
Recovery Study
Lanthanum
Europium
Terbium
Erbium
100%
Precipitation of Phosphoproteins by
Trivalent Europium-, Terbium- and Erbium- Ions
Recovery Study
ccasein = 300µg/ml cprecip. = 2M
79
Precipitation of Phosphopeptides by
Trivalent Lanthanide Ions
A Bottum-Up Approach
80
A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
Workflow for the precipitation of phosphorylated peptides 81
A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
MALDI mass spectra taken from peptide mixture (α-casein, β-casein and ovalbumin) 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+. Phosphorylated peptides are labeled.
Erbium
Holmium
Cer
82
A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
MALDI mass spectra taken from peptide mixture (α-casein, β-casein and ovalbumin) after precipitation with trivalent lanthanide ions. A, phosphopeptide enriched by precipitation with La3+. B, phosphopeptide enriched by precipitation using Eu3+. C, phosphopeptide enriched by precipitation using Tm3+. Only phosphorylated peptides are labeled
Lanthanum
Europium
Terbium
83
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-casein respectively. β-C refers to peptides form β-casein
Erbium
Holmium
Cer
84
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
85
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
86
[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
Tetra
-
-
-
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
-
-
+
-
+
-
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
-
-
+
+
-
+
+
+
-
+
+
-
-
-
+
+
+
+
-
+
+
+
+
+
-
+
+
-
+
+
-
-
+
+
-
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
+
-
+
-
+
-
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
-
+
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
+
+
+
+
-
+
+
+
+
-
-
-
-
-
+
+
-
-
-
-
-
+
+
-
+
+
-
-
-
+
+
+
+
-
+
+
+
+
-
-
+
-
+
+
+
-
+
-
+
-
+
-
+
+
+
Recovery of Phosphopeptides
23 20 19 20 21 12 14 18
P P
P
P P
3.6% HCl
P P
P P
P
trypsin
Tryptic On-Pellet Digest of Precipitated Phosphoproteins
Inte
nsity
m/z 88
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
P P
P
P P
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 CeCl3 1466.6 153-164 α-S2 TVDMESpTEVFTK mono + + - + + + + +
1482.6 153-164 α-S2 TVDM*ESpTEVFTK mono + + - - + - + +
1594.7 153-165 α-S2 TVDMESpTEVFTKK mono + + + + + + + +
1610.7 153-165 α-S2 TVDM*ESpTEVFTKK mono + + - - + - + +
1660.7 121-134 α-S1 VPQLEIVPNSAEER mono + + + + + + + +
1832.8 104-119 α-S1 YLGEYLIVPNSpAEER mono + + + + + + + +
1847.6 58-73 α-S1 DIGSESpTEDQAMEDIK mono + + - - - - - -
1927.6 58-73 α-S1 DIGSpESpTEDQAMEDIK di + + - + + + + +
1943.6 58-73 α-S1 DIGSpESpTEDQAM*EDIK di + + + + + + + +
1951.9 119-134 α-S1 YKVPQLEIVPNSpAEER mono + + + + + + + +
1981.8 48-63 β FQSpEEQQQTEDELQDK mono + + + + - + + -
2000.0 118-134 α-S1 KYKVPQLEIVPNSpAEER mono + + - + + - - +
2061.8 48-63 β FQSpEEQQQTEDELQDK mono + + + + + + - -
2080.0 118-134 α-S1 KYKVPQLEIVPNSpAEER mono + + + + + + + +
2432.0 45-63 β IEKFQSpEEQQQTEDELQDK mono + + - + + - - -
2548.2 119-139 α-S1 YKVPQLEIVPNSpAEERLHSMK mono + + - + + - + +
2560.1 44-63 β KIEKFQSpEEQQQTEDELQDK mono + + - + + - - -
2564.6 119-139 α-S1 YKVPQLEIVPNSpAEERLHSM*K mono + + - + + - + +
2598.0 52-73 α-S1 VNELSpKDIGSpESpTEDQAMEDIK di + + - + + - + +
2678.0 52-73 α-S1 VNELSpKDIGSpESpTEDQAMEDIK tri + + - + - + + +
2703.5 114-135 α-S1 LRLKKYKVPQLEIVPNSpAEERL mono + + - + + + - -
2716.2 130-152 α-S2 NAVPITPTLNREQLSpTSpEENSKK di + + - + + - + +
2720.9 74-94 α-S1 QMEAESpISpSpSpEEIVPNSpVEQK penta + + - + + - - -
2747.1 17-37 α-S2 NTMEHVSpSpSpEESpIISQETYKQ tetra + + + + + + - -
2856.5 50-73 α-S1 EKVNELSpKDIGSESTEDQAMEDIK di + + - + + - + +
2935.1 50-73 α-S1 EKVNELSpKDIGSpESTEDQAMEDIK tri + + - + - - + +
2966.1 17-40 β ELEELNVPGEIVESpLSpSpSpEESITR tetra + + + + + - - -
3008.0 61-85 α-S2 NANEEEYSIGSpSpSpEESpAEVATEEVK tetra + + + + + + + +
3024.2 16-40 β RELEELNVPGEIVESLSpSpSpEESITR tri + + + + + - - -
3042.2 16-40 β RELEELNVPGEIVESLSpSpSpEESITR tri + + + + + - - -
3087.9 61-87 α-S2 NANEEEYSpIGSpSpSpEESpAEVATEEVK penta + + - + + - - -
3122.2 16-40 β RELEELNVPGEIVESpLSpSpSpEESITR tetra + + + + + + + +
3132.2 16-39 α-S2 KNTMEHVSpSpSpEESpIISQETYKQEK tetra + + - + + + + +
34 15 31 16 22
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 fraction CeCl3
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
92
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
Gelelektrophorese
Structure of the Repeating Unit of Agarose, 3,6-anhydro-L-galactose
Basic disaccharide
repeating units of
agarose,
G: 1,3-β-d-galactose
and
A: 1,4-α-l-3,6-
anhydrogalactose
Gel Structure of Agarose
Crosslinking Acrylamide Chains
Improvements in 2D-PAGE
- +
- IPG (Immobilised Ph Gradient) strips for the first dimension
pH-forming chemical groups are grafted onto the polyacrylamide matrix, creating a mechanically stable pH gradient
+ + mechanical stability
+ + reproducibility
+ + loadable amounts
+ + „zoom“ pI ranges
pH 3.0 10.0
1st dimension
Protein sample can be applied at sample application well following
the rehydration step if the protein sample was not included in the
rehydration solution.
Place assembled strip holder on electrophoresis platform
Equilibration step 1 :
• SDS
• Buffer pH 6.8
• DTT (reduce –S-S-)
• Iodoacetamide
(alkylate –S-S-)
After IEF : equilibration and 2nd dimension
+ -
6.5 10 3.0 5.5 8.5
pI
100
75
50
37
20
10
150
kDa
Size
horizontal gel electrophoresis
Electrophoresis Equipment
Vertical gel
Electrophoresis Equipment
Combs are used to put wells in the cast gel for sample loading.
– Regular comb: wells separated by an “ear” of gel
– Houndstooth comb: wells immediately adjacent