Large-Scale Peptide Synthesis & Purification

Jacob et al. (2004) EP 04 012 691.4 Jacob et al. (2004) EP 04 012 720.1 Dauber et al. (2010), submitted.

Elution of shorter derivatives

TFA-cleavage

Simultaneous removal of fmoc- group and elution of purified products

Transfer of unpurified fmoc-protected products into plate containing C18 material

TfiC

Automated synthesis in filter-bottom microtiter plates

Synthesis at Rink resin

Mass spectrometry

ETS-1: Human erythroblastosis virus oncogene homolog 1 (HUMETS1A) => 62 peptides

AP-2: DNA-binding protein (HUMAP2) => 61 peptides

KLF-5: Kruppel-like factor 5 (AF287272)

Akt: Protein kinase B, gamma (AL592151)

NFAT: Nuclear factor of activated T-cells (BC001050)

Brn-3b: Human POU domain protein (HSU06233)

Y-Box: Y box binding protein 1 (BC098435) => 45 peptides

NF-kappaB: NF-kappaB repressing factor (BC068514) => 136 peptides

Transcription Factors Related to Breast Cancer

positional control buffer

CxPTADGPYLQILEQPKQRG CxYLQILEQPKQRG FRFRYV

CxQRG FRFRYVCEGPSHGGL CxCEGPSHGGLPGASSEKNK CxGGLPGASSEKNKKSYPQV

proteins proteins

buffer positional control positional control

Aminoacid consensus sequence of NF-kappaB

0.1 1.0 10.0 100.0

PCR template [pg]

Exp

ress

ed p

rote

in [n

g/µl

]

300

200

100

In Situ Protein Synthesis Synthetic Biology

Angenendt et al. (2006) Mol. Cell. Prot. 5, 1658-1666.

spotting spotting transcription rehydration & washing & DNA template & translation mixture incubation detection

In Situ Protein Synthesis Synthetic Biology

Direct detection of GFP at 488nm

Green Fluorescence Protein

spotting spotting transcription rehydration & washing & DNA template & translation mixture incubation detection

F R •  Forward and reverse primer at surface

Presentation of an Individual Proteome Synthetic Biology

•  Annealing of template RNA (or DNA) •  Extension of forward primer (reverse transcription)

•  Removal of template

•  Detection of extension reaction

•  Initial PCR

•  Subsequent amplification

spotting spotting transcription rehydration & washing & DNA template & translation mixture incubation detection

Protein Expression & Structural Profiling Antibody Microarrays

EU

Goat anti-mouse IgG Goat anti-rabbit IgG

Dual colour incubation with two serum samples

Alhamdani et al. (2010) J. Prot. Res. 9, 963-971. Gloriam et al. (2010) Mol. Cell. Prot. 9, 1-10. Schröder et al. (2010) Mol. Cell. Prot. 9, 1271-1280. Alhamdani et al. (2010) Proteomics, 10, 3203-3207. Schmidt et al. (2011) J. Prot. Res. 10, 1316-1322. Schröder et al. (2011) Antibody Engineer., in press. Mustafa et al. (2011) Mol. Biosyst. 7, 1795-1801.

Goat anti-mouse IgG Goat anti-rabbit IgG

Protein labelling with Sypro Ruby

Positional marker proteins

Human serum samples

Human urine samples

Current Chip Layout Antibody Microarrays

810 different antibodies 678 selected based on significant variations in transcriptional studies 67 additional keyplayers in cancer-related pathways 40 from literature research and collaboration partners 25 housekeeping proteins and controls

Selection of Antibodies

Transcriptional Profiling

Expression Profiling

•  DNA sequences of 900 genes/ESTs exhibiting differential transcription

•  Peptide selection from sequences according to 12 selection criteria

•  Peptide synthesis

•  Immunisation of rabbits

•  Affinity purification of polyclonal antibodies

•  Characterisation by in situ analyses

Antibody Microarrays

Efficiency for Particular Protein Markers

Alhamdani et al. (2010) J. Prot. Res. 9, 963-971.

Mix1 Mix2 Q-Prot

Catalase

Per

oxis

ome

Mix1 Mix2 Q-Prot

TGN-46

Golgi apparatus

Mix1 Mix2 Q-Prot

Calrectinulin

ER

Mix1 Mix2 Q-Prot

Lamin

Nucleus

Mix1 Mix2 Q-Prot

Cytochrome C

Mitochondrion

Mix1 Mix2 Q-Prot

Actinin

Cytoskeleton

Mix1 Mix2 Q-Prot

Flotilin

Pla

sma

mem

bran

e

gamma-GT

Mix1 Mix2 Q-Prot

Lipid and Dye Removal Antibody Microarrays

Alhamdani et al. (2010) Proteomics 10, 3203-3207.

Surface Blocking Antibody Microarrays

Ratio DY-649/ DY-549

DY-649

DY-549

Ctrl 1% EA 3% 5% 10% 2% 1% EA Candor 5% 10% PBST + 3% BSA BSA BSA BSA globulin + SDS Milk Milk

Alhamdani et al. (2010) Proteomics 10, 3203-3207.

•  Production of 1080 microarrays of 810 antibodies each in five consecutive batches from same set of source plates.

•  Per antibody, 10 μg are needed.

•  Optimisation of production for high intra- and inter-array homogeneity.

•  Incubation of 20 arrays with the same samples: 2 Arrays from the start and the end of each batch, respectively.

•  Coefficient of variation (CV) in all production is 13% on average.

•  CV < 15% for 89% of all features. •  CV < 20% for 96% of all features.

Production Quality Antibody Microarrays

Schröder et al. (2010) Mol. Cell. Prot. 9, 1271-1280.

Analysis of urine samples: •  healthy males •  healthy females

Profiling Pancreatic Adenocarcinoma Antibody Microarrays

•  diseased males •  diseased females

Male & normal

Male & tumour

Female & normal

Female & tumour

Males Females

Tum

our

Nor

mal

Schröder et al. (2010) Mol. Cell. Prot. 9, 1271-1280.

> 500 total RNA samples

> 700 protein samples

> 1000 DNA-samples

> 700 Histochemical analyses

•  Epigenetic analysis •  Mutation analysis •  SNP-typing

•  DNA-microarrays > 500 mRNA analyses > 250 miR analyses

•  Antibody-microarrays •  Protein-microarrays

Splitting in 3 homogenous fractions; separate isolation

Mixing slices

> 350 blood & urine samples

Studying Pancreatic Cancer

Material from 1200 patients tumours T1-T4, cystic tumours, pancreatitis and normal.

Frozen Cryotome tumour tissues 10 µm slices

DropTop & ARTAMIS Consortia

EU

Parallel dissociation analysis

Quantitative Analysis