6 Materials and Methods

Whatman Inc. 27 Great West Road Brentford, Middlesex TW8 9BW, United Kingdom GE Healtcare München Germany Pierce Inc. Rockford, IL USA WTW GmbH Dr.-Ka...

0 downloads 229 Views 200KB Size
Materials and Methods

6 Materials and Methods 6.1 Materials and Instrumentation 6.1.1 Proteins and Peptides Angiotensin I Angiotensin II Bovine serum albumine (BSA) Peptide Calibration Standard Calmodulin, high purity (Bos taurus, brain) Carbonic Anhydrase EC 4.2.1.1 Chymotrypsin (EC 3.4.21.1, Bos taurus), sequencing grade Cytochrome c (Gallus gallus) Endoproteinase AspN (EC 3.4.21.33, Pseudomonas fragi mutant), sequencing grade

Sigma Sigma Sigma Bruker Daltonik Calbiochem Sigma Roche Diagnostics Sigma Roche Diagnostics

Endoproteinase LysC (EC 3.4.21.50, Lysobacter enzymogenes), sequencing grade

Roche Diagnostics

Endoproteinase GluC (EC 3.4.21.19, Staphylococcus aureus V8) sequencing grade

Roche Diagnostics

Immunoglobulin A Lactate dehydrogenase Luteinizing hormone releasing hormone (LHRH) Lysozyme, chicken egg white Melittin, synthetic Monoclonal Anti-Annexin II, Clone CPI-50-5-1 (Mus musculus) Myoglobin (Equus caballus ,heart) Phosphorylase b Somatostatin Substance P Trypsin (EC 3.4.21.4, Bos taurus), sequencing grade

Sigma Sigma Sigma Sigma Calbiochem ICN Biomedicals Sigma Sigma Sigma Sigma Roche Diagnostics

6.1.2 Cross-Linking and Labeling Reagents (+)-Biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-iodoacetyl biotin) Bis(sulfosuccinimidyl) glutarate (BS2G), d0 and d4 Bis(sulfosuccinimidyl) suberate (BS3), d0 and d4 Disuccinimidyl adipate (DSA), d0 and d8 Disulfosuccinimidyl tartrate (sDST) Ethylene glycol bis(succiimidly succinate) (EGS) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) N-Hydroxysulfosuccinimide (Sulfo-NHS) N-succinimidyl-6-[4’-azido-2’-nitrophenylamino]hexanoate (SANPAH)

Pierce Pierce Pierce K. Mechtler, IMP, Vienna Pierce Pierce Pierce Pierce Pierce

104

Materials and Methods

6.1.3 Chemicals Acetic acid, glacial Acetone, Uvasol® for spectroscopy Acetonitrile (ACN), Uvasol® for spectroscopy Acrylamide/Bis solution 40% (37.5:1) α-Cyano-4-hydroxy cinnamic acid Ammonium acetate Ammonium hydrogencarbonate (NH4HCO3) Ammonium persulfate (APS) 6-aza-2-thiothymine (ATT) Calcium chloride Complete, EDTA-free protease inhibitors Coomassie-Brilliant-Blue R250 and G250 DEAE, pre-swollen microgranular DEAE cellulose (DE52) 2’,5’-Dihydroxyacetophenone 2,5-Dihydroxybenzoic acid (DHB) 3,5-Dimethoxy-4-hydroxy cinammic acid (sinapinic acid) Dimethyl sulfoxide (DMSO) Dithiothreitol (DTT) E-64 (N-[N-(L-3-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine) Ethylene glycol tetraacetic acid (EGTA) Formic acid (FA) GelCode® Glycoprotein Staining Kit Glycine Hydrochloric acid (HCl) 4-Hydroxyazobenzene-2-carboxylic acid (HABA) Imidazole Iodoacetamide N-(2-Hydroxyethyl)piperazin-N´-(2-ethansulfonsäure) (HEPES) Isopropanol, Uvasol®, for spectroscopy 2-Mercaptoethanol Methanol, Uvasol®, for spectroscopy 2-(N-Morpholino)ethane sulfonic acid (MES) Native Sample Buffer, Laemmli PeppermintStick™ phosphoprotein molecular weight standard Ponceau S Potassium chloride ProQ ® Diamond phosphoprotein gel stain Precision Plus Protein™ Unstained Standards (10-250 kDa) Sample Buffer, Laemmli sodium dodecyl sulfate (SDS) solution, 10% Sodium chloride N,N,N´,N´-Tetramethylethylendiamine (TEMED) Triluoro acetic acid (TFA) Tris / Glycine Running buffer (10x) Trishydroxymethylaminomethane (Tris-Base) Trishydroxymethylaminomethane hydrochloride (Tris-HCl) Triton X-100 (octylphenolepoly(ethyleneglycolether)X) UltraLink Immobilized Monomeric Avidin

Merck/VWR Merck/VWR Merck/VWR Bio-Rad Bruker Daltonik, Sigma Sigma Sigma Bio-Rad, Sigma Sigma Sigma Roche Diagnostics Sigma Whatman Sigma Sigma Sigma Sigma Sigma Roche Diagnostics Sigma Sigma, Merck/VWR Pierce Sigma Sigma Sigma Sigma Sigma Sigma Merck/VWR Bio-Rad Merck/VWR Sigma Bio-Rad Invitrogen/Molecular Probes Sigma Invitrogen/Molecular Probes Bio-Rad Bio-Rad Bio-Rad Sigma, Roth Bio-Rad Sigma Roth Sigma Sigma Pierce

105

Materials and Methods

6.1.4 Instrumentation MALDI-TOF Mass Spectrometers: Voyager-DE™ RP Biospectrometry™ Workstation (Applied Biosystems) Autoflex I (Bruker Daltonik) Ultraflex III (Bruker Daltonik) ESI-FTICRMS: Apex II (Bruker Daltonics), 7T magnet, Nano-ESI source (Agilent) LTQ-FT (ThermoScientific), 7T magnet, Nano-ESI source (Proxeon) Nano-High-Performance Liquid Chromatography: Ultimate™ Nano-HPLC System, (LC Packings / Dionex) equipped with: Ultimate™ Micropump Ultimate™ UV-Detector Ultimate™ SWITCHOS II Famos™ Micro-Autosampler Ultimate™ 3000 Nano-HPLC System, (LC Packings / Dionex) Fast Protein Liquid Chromatography (FPLC): ÄKTA Explorer (GE Healthcare)

6.1.5 Miscellaneous Equipment and Consumables Analytical balances

OHAUS Adventurer ARA520 OHAUS Adventurer ARA640

OHAUS

Bench-top shakers

Duomax 1030 Titramax 101

Heidolph

Bench-top centrifuge

MiniSpin

Eppendorf

Centrifuge

Avanti J-20 XP, rotor JLA 16.250 Optima LE-80K ultracentrifuge Microcon YM-3 and YM-10 centrifugal filter device (3 and 10 kDa cut-off) ZipTips, C4 and C18 Centriprep YM-30, centrifugal filter device (30 kDa cut-off) Dialysis membranes Spectra/Por®

Beckman Coulter

Electrophoresis

Mini-PROTEAN 3 cell, POWERPAC 300 power supply, glass plates with integrated spacer, short plates, side-by-side casting stand, casting frames, electrode, clamping frame, sample loading guide, square-bottom plastic combs

Bio-Rad

Heating and drying oven Imaging

Heraeus T6 Function Line Gel Image Scanner PharosFX Molecular Imager System

Thermo Fisher Amersham Bio-Rad

Desalting / Buffer Exchange

Millipore Millipore Millipore Roth

106

Materials and Methods

pH-Meter

inoLab pH Level 1

WTW

Protein purification

Potter S homogenizer, homogenizer vessel, homogenizer cylinder, plunger Waring laboratory blender

Sartorius

Vacuum concentrator

Concentrator 5301 (SpeedVac)

Eppendorf

Water

DirectQ5TM water purification system

Millipore

Western Blot equipment

Trans-Blot Semi-Dry cell

Bio-Rad

Waring

6.1.6 Software ASAP

Automatic Spectrum Assignment Program, software for assigning MS peaklists generated from chemical cross-linking experiments, available at http://roswell.ca.sandia.gov/~mmyoung/asap.html

DataExplorer v. 4.0

Software for processing MALDI-TOF mass spectra (Applied Biosystems) Analytical tools for identification, sequence analysis, and tertiary structure prediction of proteins; database search; www.expasy.org

ExPASy Proteomics Server

flexControl v. 2.2.19.0

Acquisition software for MALDI-TOFMS (Bruker Daltonik)

flexAnalysis v. 2.2

Software for processing of MALDI-TOF mass spectra (Bruker Daltonik)

GETAREA 1.1

Software that calculates the solvent accessible surface area of molecules; www.chem.ac.ru/Chemistry/Soft/GETAREA.en.html

GPMAW v. 7.01 and below

General Protein/Mass Analysis for Windows (Lighthouse Data, www.welcome.to/gpmaw); evaluation of peaklists from MS analysis for identification of cross-linked products and peptides

IsoFind

In-house developed tool for searching for distinct distances between two signals from a given MS peaklist and for calculating signal ratios based on signal intensity or peak area

Mammoth

Matching Molecular Models Obtained from Theory, program for sequence independent structure alignment of proteins

Mascot

Performs peptide mass fingerprint analysis from a given MS peaklist, www.matrixscience.com

Mascot Distiller v. 1.1, 2.0

Software for processing mass spectra (Matrix Science)

MS2 Assign

Tool for assigning peaklists from MS/MS experiments to a theoretical fragment library for cross-linked products, modified peptides, and unmodified peptides available at http://roswell.ca.sandia.gov/~mmyoung/ms2assign.html

Profound

Performs peptide mass fingerprint analysis for a given MS peaklist

Rasmol v. 2.7.3

Visualization and analysis of protein structures, www.openrasmol.org

Rosetta

Program for modeling, docking, etc of proteins; www. rosettacommons.org

107

Materials and Methods

UMAX scanner software

Operates UMAX scanner

Unicorn v. 4.10

Software for operating ÄKTA FPLC systems (Amersham Biosciences)

Voyager v. 5.1

Acquisition software for MALDI-TOFMS (Applied Biosystems)

VMD-Explorer v. 1.8.1

Visualization and analysis of protein structures (Theoretical and Computational Biophysics Group, NIH, www.ks.uiuc.edu)

XMASS vs, 5.0.10, 6.0 and 7.02

Software for acquisition and processing of ESI-FTICR mass spectra (Bruker Daltonics)

Xplor-NIH

Structure determination program, http://nmr.cit.nih.gov/xplor-nih/

6.1.7 List of Manufacturers Agilent Technologies Waldbronn Germany

ICN Biomedicals, GmbH Mühlgrabenstr. 12 53340 Meckenheim

Roche Diagnostics GmbH Sandhofer Str. 116 68305 Mannheim, Germany

Applied Biosystems 850 Lincoln Drive Foster City, CA 94404, USA

Invitrogen / Molecular Probes 3 Fountain Drive Inchinnan Buisness Park Paisley PA4 9RF, UK

Roth (Carl-Roth GmbH & Co. KG) Schoemperlenstr. 3-5 76185 Karlsruhe, Germany

Bio-Rad 1000 Alfred Nobel Drive Hercules, CA 94547, USA

LC Packings / Dionex Amsterdam The Netherlands

Sartorius AG Weender Landstr. 94-108 37075 Göttingen, Germany

Bruker Daltonics Billerica, MA USA

Lighthouse Data Odense Denmark

Sigma-Aldrich Chemie GmbH Eschenstr. 5 82024 Taufkirchen, Germany

Bruker Daltonik GmbH Bremen Germany

Merck / VWR Darmstadt Germany

Thermo Electron Bremen Germany

Calbiochem Schwalbach am Taunus Germany

Millipore Eschborn Germany

Waring Laboratory and Science waringproducts.com

Eppendorf GmbH Peter-Henlein-Str. 2 50389 Wesseling-Berzendorf Germany

New Objective Woburn, MA USA

Whatman Inc. 27 Great West Road Brentford, Middlesex TW8 9BW, United Kingdom

GE Healtcare München Germany

Pierce Inc. Rockford, IL USA

WTW GmbH Dr.-Karl-Slevogt-Strasse 1 D-82362 Weilheim

Heidolph Instruments GmbH & Co. KG Walpersdorfer Str. 12 91126 Schwabach, Germany

Proxeon Biosystems Odense Denmark

108

Materials and Methods

6.2 Experimental Procedures 6.2.1

Isolation and Purification of A2t

The ANXA2 / p11 heterotetramer (A2t) was purified from mucosa of pig (Sus scrofa) small intestines following a slightly modified version of the protocol of Gerke and Weber (1984). Six fresh small intestines were obtained from the slaughterhouse Altenburg and processed immediately after evisceration. The small intestines were washed with 30 liters ice-cold imidazole buffer (10mM imidazole, 150 mM NaCl, pH 7.4) to remove the gut contents and were cut into pieces of about 50 cm length and were slit lengthwise. The thin mucosal layer was scraped off and was immediately frozen in liquid nitrogen for storage. Henceforth, all steps of the purification procedure were carried out at 4°C. Back in the laboratory, the frozen material was thawed, 1.5 l HEPES/Tris buffer (30 mM HEPES, 600 mM NaCl, 0.5% Triton X-100, 1 mM CaCl2, pH 7.4, Tris was used for adjusting pH) was added, and the mucosa was mechanically disrupted using a Waring blender. In addition to protease inhibitors, the buffer contained 1 mM Ca2+, which causes A2t binding to the membrane. The calcium concentration was raised to 2 mM and the homogenate was stirred for 15 minutes. After a centrifugation step (34,000 x g, 60 minutes, Avanti J-20 XP centrifuge, JLA 16.250 rotor, 4°C) the supernatant was discarded and the pellet containing A2t was retained. In total, the sample was washed and centrifuged three times with the above mentioned buffer containing the detergent Triton X-100, and was washed another three times without detergent. In a next step, the pellet was resuspended in 300 ml HEPES/Tris buffer (30 mM HEPES, 600 mM KCl) containing 10 mM ethylene glycol tetraacetic acid (EGTA) and the cells were thoroughly disrupted in a Potter homogenizer (Sartorius). Complexation of Ca2+ by EGTA released A2t from the membrane so that after ultracentrifugation (45,000 rpm, 60 minutes, Optima LE-80K ultracentrifuge, 4°C) the A2tcontaining supernatant was retained. For preparation for DEAE (diethylaminoethyl cellulose, Whatman) anionic exchange chromatography, the supernatant was dialyzed over-night against two-times 15 l 20 mM imidazole / 10 mM NaCl / 0.5 mM EGTA buffer (pH 7.5). The dialyzed protein solution was applied onto the equilibrated DEAE column (column volume approx. 70 ml) and A2t was obtained in the flow-through. The obtained A2t was used without further purification. Chromatographic separation steps were conducted with the ÄKTA Explorer fast protein liquid chromatography (FPLC) system (GE Healthcare). The flow rate was manually adjusted and a one-step gradient of 10 mM and 1M NaCl was applied. UV absorption at 280 nm and conductivity were monitored. Protease inhibitors Complete EDTA-free and E-64 as well as dithiothreitol (DTT, 1mM) were applied during the whole course of the purification. After each purification step aliquots were taken for monitoring the purification process and were stored at –20°C before SDS-PAGE and MS analyses were performed. 109

Materials and Methods

The purified A2t was concentrated using Centripreps YM-30 (Millipore, 30 kDa cut-off) and the buffer was at the same time exchanged against 20 mM HEPES / 150 mM NaCl / 0.5 mM EGTA, 0.5 mM DTT (pH 7.4). The purified A2t was then lyophilized for storage at –20°C.

6.2.2

Characterization of A2t

Purification of A2t was confirmed by Western blot analysis and A2t was characterized with respect to amino acid sequence and possibly existing posttranslational modifications. 6.2.2.1

Western Blot Analysis

Unstained gels from SDS-PAGE (chapter 6.2.6.2) were equilibrated in blot buffer (48 mM Tris, 39 mM glycine, 1.3 mM SDS, 20 % methanol (v/v), pH 9.2) for 20 minutes. Nitrocellulose membranes were cut to gel size and placed for three minutes in blot buffer. Filter paper was as well soaked in blot buffer. A sandwich composed of wet filter paper, nitrocellulose membrane, equilibrated gel, and another wet filter paper was placed on the bottom platinum anode of the TransBlot® Semi-Dry Transfer Cell (Bio-Rad) and the stainless steel cathode and safety cover were placed on top. Transfer of the proteins from the gel onto the nitrocellulose membrane was conducted at 10V (PowerPack300, Bio-Rad) for 30 minutes. Afterwards, the membrane was incubated in fixing solution (40 % methanol (v/v), 10 % trichloro acetic acid (TCA) (v/v)) for 20 minutes. Ponceau S staining was employed for confirming the protein transfer to the membrane. For blocking the membrane a 1% (w/v) bovine serum albumin solution in TTBS (Tween 20 (0.05% (v/v)) in TBS (20 mM Tris, 150 mM NaCl, pH 7.5)) was used, in which the membrane was incubated for 90 minutes under gentle agitation. The BSA / TTBS solution was discarded. Monoclonal anti-annexin II (clone CPI-50-5-1, 0.25 mg/ml, from Mus musculus, ICN Biomedicals) was diluted 1:5000 in TTBS and used for incubating the membrane (60 minutes). Unbound primary antibody was removed by three consecutive two-minute washing steps with TTBS and replaced by the secondary antibody anti-mouse IgG (diluted 1:10000 in TTBS, from goat (Capra hircus), Sigma product no. A3562)). This mouse-specific antibody is linked to the reporter enzyme alkaline phosphatase (AP). Incubation time was 45 minutes after which excess anti-body was again removed by two washing steps with TTBS and one washing step with 1x Development Buffer (Bio-Rad, AP Color Development kit). For colorimetric detection, 300 µl each of AP Color Reagents A (nitroblue tetrazolium, NBT) and B (5-bromo-4-cloro-3-indolyl phosphate, BCIP) (Bio-Rad) were mixed with 30 ml 1x Development Buffer. The nitrocellulose membrane was immersed in the color development solution for three minutes under gentle agitation. The reaction of BCIP with alkaline phosphatase results in a blue precipitate that is converted by NBT to result in purple color. Then the membrane was thoroughly rinsed with water and the air-dried membrane was imaged for documentation. 110

Materials and Methods

6.2.2.2

Amino Acid Sequences

The amino acid sequence of porcine ANXA2 was determined by ESI-FTICRMS (chapter 6.2.8.2) and MALDI-TOFMS (Autoflex I, chapter 6.2.7.3) of enzymatic digests (chapter 6.2.6.1). The obtained MS peaklists were compared to Swiss-Prot database (www.expasy.org) entries available for Homo sapiens (P07355), Bos taurus (P04272), Canis familiaris (Q6TEQ7), Sus scrofa (P19620, fragment of amino acids 1-91), and porcine amino acid sequences suggested by V. Gerke (unpublished) and M. (Ph.D. thesis, 2005). ANXA2 amino acid sequence assessment was performed using the Expasy Find Pept tool (chapter 6.2.11.3). Experimentally obtained p11 peptide masses were compared to Swiss-Prot entry P04163. 6.2.2.3

Molecular Weight Determination of ANXA2 and P11

Linear MALDI-TOFMS analysis (Autoflex I) in the positive ionization mode of the purified A2t complex was performed for determination of the molecular weights of ANXA2 and p11 (chapter 6.2.7.3). Several MALDI matrices (sinapinic acid, DHB, ATT, HABA, and acetophenone) were tested for obtaining optimum mass spectra of ANXA2. 6.2.2.4

Posttranslational Modifications

Phosphorylation Analysis ANXA2 and p11 were tested for phosphorylation using the Pro-Q® Diamond Phosphoprotein Gel Stain Kit (Invitrogen/Molecular Probes), an in-gel fluorescent detection assay for phosphorylation of Tyr, Ser, and Thr. PeppermintStick™ phosphoprotein molecular weight (MW) standard contains a mixture of two phosphorylated (ovalbumin (45 kDa) and α-casein (23.6 kDa)) and four non-phosphorylated proteins (β-galactosidase (116.25 kDa), BSA (66.6 kDa), avidin (18 kDa), and lysozyme (14.4 kDa)), thus functioning both as molecular weight standard and as positive and negative controls. Following separation of A2t (20 µl of a 5.5 µM solution) and the MW standard by gel electrophoresis, without subsequent Coomassie Brilliant Blue-staining, the gel (5% stacking / 12% resolving) was incubated twice in fixing solution (50% (v/v) methanol / 10% (v/v) acetic acid) for 30 minutes. The fixing solution was removed and the gel was immersed in H2O for 10 minutes. Washing with H2O was repeated twice for thorough removal of residual methanol and acetic acid. The gel was then incubated in ~ 60 ml of the staining solution for 90 minutes in the dark. Afterwards, the gel was destained in 100 ml destaining solution for 30 minutes, again protected from light. Destaining was repeated two more times with fresh solution and afterwards the gel was thoroughly immersed in H2O. Visualization of stained protein bands was achieved with the PharosFX Molecular Imager System (Bio-Rad). The excitation and emission maxima of the Pro-Q® Diamond stain are ~555

111

Materials and Methods

nm and ~580 nm, respectively. Following documentation, the gel was stained with Coomassie Brilliant Blue.

Glycosylation Analysis ANXA2 and p11 were tested for glycosylation using the GelCode® Glycoprotein Staining Kit (Pierce). Horseradish peroxidase and soybean trypsin inhibitor served as positive and negative controls, respectively. Gels from electrophoretic separation of A2t, positive and negative controls, and MW standard were fixed by 50% (v/v) methanol for 30 minutes. The solution was replaced by 100 ml 3% (v/v) acetic acid and incubated for ten minutes. This washing step was repeated once more, before the gel was transferred to 25 ml of Oxidizing Solution (periodic acid) and was incubated for 15 minutes, oxidizing glycols to aldehydes. The gel was washed three times for five minutes with 100 ml of 3% (v/v) acetic acid and was then immersed in 25 ml of GelCode® Glycoprotein Stain for 15 minutes. Afterwards the gel was transferred into 25 ml Reducing Solution and incubated for five minutes, after which the gel was extensively washed with 3% (v/v) acetic acid and H2O. Glycoproteins appeared as magenta bands on the gel. The GelCode® Glycoprotein Staining Kit reagents were prepared as specified by the manufacturer.

6.2.3 Chemical Cross-Linking 6.2.3.1

Cross-Linking Reactions of the Calmodulin / Melittin Complex

For chemical cross-linking of the calmodulin / melittin complex the zero-length cross-linking reagent 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in combination with sulfo-N-hydroxysuccinimide (sNHS) was employed, as well the homobifunctional amine-reactive reagents sulfo-disuccinimidyl tartrate (sDST) and bis-sulfosuccinimidyl suberate (BS3). EDC / Sulfo-NHS An equimolar mixture of CaM and melittin (10µM each, final concentration) containing 1mM CaCl2 (in 100mM MES buffer, pH 6.5) was incubated at room temperature for 20 min on a shaker. The zero-length cross-linker EDC was dissolved in water shortly prior to addition and added in 500-, 1000-, and 2000-fold molar excess over the protein/peptide mixture. Simultaneously, sNHS, dissolved in water, was added in 500-fold molar excess to all three EDC-containing reaction mixtures, thus resulting in EDC/sNHS ratios of 1:1, 2:1, and 4:1, respectively. As a negative control, the protein mixture was incubated without cross-linking reagent. The final volume was 1 ml. For quenching the reactions, 200µl-aliquots were taken from the reaction mixtures after 5, 15, 30, 60, and 120 min, and DTT (40mM final concentration) was added and the samples were stored at –20°C.

112

Materials and Methods

Table 6-1: Composition of the CaM / Mel Reaction Mixture with the Cross-Linker EDC / sNHS I

II

III

IV

-

1:1

2:1

4:1

0.4M EDC*

0µl

12.5µl

25µl

50µl

I: 0; II: 5mM; III: 10mM; IV: 20mM

0.4M sNHS*

0µl

12.5µl

12.5µl

12.5µl

I: 0; II, III, IV: 5mM

1mg/ml Melittin**

28.5µl

28.5µl

28.5µl

28.5µl

10µM

1mg/ml Calmodulin**

167µl

167µl

167µl

167µl

10µM

794.5µl

769.5µl

757µl

732µl

≈ 0.1M

10 µl

10 µl

10 µl

10 µl

1mM Ca2+

EDC/Sulfo-NHS ratio

0.1M MES (pH 6.5) 100mM CaCl2**

Final concentration

* in H2O, ** in MES buffer

sDST and BS3 For cross-linking experiments with the homobifunctional cross-linking reagents BS3 or sDST, an equimolar mixture of CaM and melittin (10µM each, final concentration) containing 1mM CaCl2 (20mM HEPES buffer, pH 7.4) was incubated at ambient temperature for 20 min. BS3 or sDST was dissolved in dimethyl sulfoxide (DMSO) to avoid hydrolysis (stock solutions: 10mM, 50mM, and 100mM, respectively) and 10µl were added to result in a 10-, 50-, and 100-fold molar excess over the proteins. The total volume of the reaction mixture was 1 ml. To quench the reactions, 200µl-aliquots were taken from the reaction mixtures after 5, 15, 30, 60, and 120 min, and NH4HCO3 (20mM final concentration) was added and the sample was stored at -20°C. Table 6-2: Composition of the CaM / Mel Reaction Mixtures with the Cross-Linkers sDST and BS3 0x 20mM HEPES (pH 7.4)

10x

784.5µl 784.5µl

50x

100x

Final conc.

784.5µl

784.5µl

20mM

100mM CaCl2**

10µl

10µl

10µl

10µl

1mM Ca2+

1mg/ml Melittin**

28.5µl

28.5µl

28.5µl

28.5µl

10µM

1mg/ml Calmodulin**

167µl

167µl

167µl

167µl

10µM

3

DMSO

10mM

50mM

100mM

10µL

10 µl

10 µl

10 µl

sDST / BS stock solution*

0, 100µM, 500µM, 1000µM

* in DMSO ** in HEPES buffer

6.2.3.2

Cross-Linking Reactions of the Annexin A2 / P11 Complex

The homobifunctional cross-linking reagents sDST, BS3, BS2G, and DSA were used for chemical cross-linking of the ANXA2 / p11 complex. The latter three reagents were applied as 1:1 mixtures of non-deuterated and deuterated species (d0/d4 or d0/d8). sDST A 5-µM solution of A2t containing 1 mM CaCl2 (100 mM MES buffer; 150 mM NaCl, 1 mM DTT, pH 7.5; final volume 120 µl) was incubated at room temperature for 10 min prior to addition of the homobifunctional cross-linker sDST. sDST was dissolved in DMSO (2 and 5 mM stock 113

Materials and Methods

solutions, prepared shortly prior to addition) and 6 µl were added to give a 20- and 50-fold molar excess over the protein concentration. For quenching the reaction, 40µl-aliquots were taken from the reaction mixtures after 15, 30, and 60 min, and NH4HCO3 (20 mM final concentration) was added. Table 6-3: Composition of the ANXA2 / P11 Reaction Mixture with the Cross-Linker sDST

20x

50x

Final conc.

A2t** 5.46 µM

110 µl

110 µl

5µM

30mM CaCl2**

4 µl

4 µl

1mM Ca2+

sDST* stock solution

2mM

5mM

added volume

6 µl

6 µl

100µM, 250µM

* in DMSO ** in MES buffer

BS3-d0/d4, BS2G-d0/d4, and DSA-d0/d8 The homobifunctional cross-linking reagents BS3, BS2G, and DSA were employed as 1:1 mixtures of their non-deuterated and deuterated form (BS3-d0/d4, BS2G-d0/d4, and DSA-d0/d8). Stock solutions (75 and 150 mM) of cross-linking reagents were prepared shortly prior to application. 6 µl were added to 594 µl of a 1.5 µM A2t solution in 20 mM HEPES, 150 mM NaCl, 1 mM DTT containing 1 mM CaCl2 (pH 7.4) to result in a 50- and 100-fold molar excess over the protein concentration (600 µl final volume). To quench the reactions, 200-µl-aliquots were taken from the reaction mixtures after 30, 60 and 120 min, and NH4HCO3 (20 mM final concentration) was added. Table 6-4: Composition of A2t Reaction Mixtures with Isotope-Labeled Cross-Linkers

50x

100x

1.5 µM

A2t 1.5µM in 20mM HEPES (pH 7.4),

594 µl

594µl

added volume

20mM 1mM Ca2+

1mM CaCl2 X-linker* stock solution

Final conc.

7.5mM

15mM

6 µl

6 µl

75 µM, 100 µM

* in DMSO

6.2.4

Identification of A2t Interaction Partners by Chemical Cross-Linking

The protocol for identification of A2t binding partners involves several steps. The first step comprises the biotinylation (and labeling with photoreactive cross-linker) of A2t. For testing biotinylation efficiency, A2t (1.5 µM, in 20mM HEPES buffer, pH 7.5) was reacted with 1, 2, and 4 mM aqueous solutions of the sulfhydryl-reactive labeling reagent (+)-biotinyl-iodoacetamidyl3,6-dioxaoctane diamine (EZ-Link PEO-iodoacetyl biotin, Pierce) for 45 and 90 minutes at 37 °C. The reaction was quenched by the addition of five-fold excess dithiothreitol (DTT) over 114

Materials and Methods

labeling reagent concentration. The extent of biotinylation was evaluated by MALDI-TOFMS of in-gel tryptic digests of modified ANXA2 and subsequent search with the ExPASy FindMod tool and the GPMAW software, with PEO iodoacetyl biotin entered as variable modification (Δm = 414.194 u). For conducting the cross-linking experiments of A2t with its binding partners, a 4mM solution PEO iodoacetyl biotin and a reaction time of 45 minutes was chosen. Two cross-linking strategies were developed for the identification of A2t interaction partners: In preparation A, A2t (6 ml, 1.5 µM, 20mM HEPES buffer, pH 7.5) was biotinylated (4mM PEO iodoacetyl biotin) and simultaneously labeled with the heterobifunctional amine- and photoreactive cross-linker N-succinimidyl-6-[4’-azido-2’-nitrophenylamino]hexanoate (SANPAH) added at a 50-fold molar excess over A2t and reacted at its amine-reactive site (45 min incubation time). In preparation B, A2t (6 ml, 1.5 µM) was only labeled with biotin (4mM solution of PEO iodoacetyl biotin). Labeling of amines was quenched by addition of Tris, and DTT (5-fold excess) was used for stopping the biotinylation reaction. In the second step, mucosal scrapings were thoroughly homogenized and washed (two times with 1 l 30 mM HEPES / 0.6 M NaCl, pH 7.5 and two times with 1 l 30 mM HEPES / 0.15 M NaCl, pH 7.5) in the presence of 1 mM CaCl2 and 1 mM DTT (supernatants were discarded). Complete EDTA-free tablets were used for protease inhibition. Then prelabeled A2t of preparation A and B (6 ml, 1.5 µM) was added to two individual mucosal preparations (~50 ml each), respectively, and allowed to interact with potential interaction partners at ambient temperature for 20 minutes. For covalent attachment of A2t with its binding partners (third step), the mixture containing preparation A (A2t labeled with amine-reacted SANPAH) was UV-irradiated for 60 minutes. Ethylene glycol bis(succinimidyl succinate) (EGS) was added to sample preparation B containing A2t labeled with PEO iodoacetyl biotin only. EGS reaction was stopped with excess Tris. For both preparations, extraction of soluble (i.e. not membrane-associated) cross-linked proteins was accomplished by thorough cell disruption, addition of EGTA (10mM) and ultracentrifugation (45,000 rpm, 45 min). The supernatant containing cytosolic biotin-labeled A2t / binding partner complexes was retained. Then SDS (0.5%) was added to the pellet for disintegrating the membrane, thus releasing membrane-associated proteins. The supernatant of the subsequent centrifugation was retained. For obtaining biotin-labeled A2t / membrane-associated protein complexes the samples were purified by affinity purification on UltraLink Immobilized Monomeric Avidin (Pierce) beads. Two-times (one each for preparation A and preparation B) 450 µl avidin beads were washed with 1.5 ml 100 mM HEPES, pH 7.5 for 10 min and then centrifuged 3 min at 5500 rpm (Eppendorf MiniSpin centrifuge). The washing procedure was repeated once with 1.5 ml 100 mM glycine, pH 2.8 and three-times 1.5 ml 100 mM HEPES, pH 7.5. Avidin beads were treated with biotin (1.5 ml 2mM biotin for 30 min). Biotin was removed by washing again one time with glycine and then with 15% MeOH in 50 mM NH4HCO3, pH 7.5. The beads were equilibrated with 1.5 ml HEPES (see above). Approximately 100 µl sample were added per 100 115

Materials and Methods

µl beads and twice the amount HEPES (30 mM, pH 7.5) was added and the mixture was incubated for 3 hours on a shaker. Afterwards the beads were centrifuged and both supernatant and beads were retained. Three consecutive washing steps with HEPES (see procedure above) removed unbound sample. Elution of affinity-purified samples was achieved by two washing steps with 50% ACN and 0.4% TFA with incubation times of 20 minutes and subsequent centrifugation. The supernatants were dried in a vacuum concentrator. The obtained biotinlabeled proteins were separated by SDS-PAGE and gel bands were excised and tryptically digested (chapter 6.2.6.1) for subsequent analysis by MALDI-TOFMS and protein identification by peptide mass fingerprinting. In addition to the Autoflex I instrument, the samples were analyzed on an Ultraflex III MALDI-TOF/TOF instrument. Tandem mass spectrometric data was obtained for most of the samples.

6.2.5

Polyacrylamide Gel Electrophoresis

One-dimensional polyacrylamide gel electrophoresis (PAGE) was carried out using the vertical Mini-PROTEAN 3 electrophoresis system with POWERPAC 300 power supply (Bio-Rad, München, Germany). SDS gels, consisting of a stacking (5%, pH 6.8) and a resolving gel (10, 12, and 15%, pH 8.8), were prepared with the ingredients listed in Table 6-5 (gel size: 8.0 cm x 7.3 cm x 0.075 cm). First, the resolving gel was prepared by combining 40% acrylamide / bisacrylamide (37.5:1, 2.6% C) solution, 1.5 M Tris-HCl (pH 8.8), sodium dodecyl sulfate (SDS), and H2O. Addition of ammonium persulfate (APS) and N,N,N’,N’-tetramethylethylenediamine (TEMED) initiated the polymerization and the mixture was immediately poured between the glass plates and overlayed with isopropanol. After 30 minutes, the gel was polymerized and the isopropanol layer was removed (rinse with water to remove residual isopropanol). The stacking gel was prepared and added on top of the resolving gel. A 10-well square-bottom comb was inserted into the stacking gel for forming the gel pockets. Table 6-5: Preparation of Polyacrylamide Gels. Stacking Gel 5% Resolving Gel X% 40% Acrylamide / Bis

640 µl

0.25 · X% = x ml

0.5M Tris-HCl, pH 6.8

1.25 ml

---

1.5M Tris-HCl, pH 8.8

---

2.5 ml

10% SDS (w/v)

50 µl

100 µl

H2O

3.025 ml

7.35 - x ml

TEMED

5 µl

5 µl

10% APS (w/v)

25 µl

50 µl

Total Volume

5 ml

10 ml

116

Materials and Methods

Native 8% (pH 8.8) gels were cast as continuous gels, i.e., without stacking gel, and SDS was replaced by H2O. For native PAGE and SDS-PAGE different sample preparation conditions, running buffers, and electrophoresis conditions were employed as described below. Following electrophoretic separation, gels were stained for 60 min with a staining solution containing 0.1% (w/v) Coomassie Brilliant Blue R-250, 50% (v/v) methanol, and 10% (v/v) glacial acetic acid. After 60 minutes the staining solution was replaced by the destaining solution (25% (v/v) methanol, 10% (v/v) glacial acetic acid) and the gels were destained until protein bands became visible and background stain was sufficiently removed. Gels were airdried between cellophane sheets for documentation. 6.2.5.1

Native Polyacrylamide Gel Electrophoresis

Native PAGE of the intact, non-cross-linked as well as cross-linked A2t was performed under non-denaturing conditions in the absence of SDS and reducing agents employing 8% continuous gels. Native sample buffer (containing 62.5 mM Tris-HCl, pH 6.8, 40% glycerol (BioRad)) without SDS and 2-mercaptoethanol was used for sample preparation in 1:1 ratio without heating and the samples were filled into the sample wells. Detergent-free, 5x concentrated running buffer (pH 8.3) containing 0.96 M glycine and 0.125 M Tris was diluted 1:5 (v/v). Electrophoretic separation was conducted at 100V for ~4 hours at 4°C. Immunoglobulin A (300 kDa), lactate dehydrogenase (140 kDa), phosphorylase b (97 kDa), bovine serum albumine (67 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12 kDa) were used as molecular weight standards. 6.2.5.2

SDS - Polyacrylamide Gel Electrophoresis

One-dimensional SDS-PAGE was conducted as described by Laemmli [Laemmli, 1970]. Samples were mixed 1:1 (v/v) with Laemmli sample buffer containing 5% (v/v) 2mercaptoethanol and heated for 5 min at 95°C. For separation of the CaM / melittin crosslinking reaction mixtures, 5% stacking / 15% resolving gels were prepared. For separation of the ANXA2 / p11 cross-linking reaction mixtures, 5% stacking and 10, 12, or 15% resolving gels were employed. Gels for monitoring the protein purification process and gels for subsequent Western Blot analysis were 5% stacking / 12% resolving gels. Running buffer (prepared from 10x concentrated running buffer containing 0.25 M Tris, 1.92 M glycine, and 1% (v/v) SDS) (Roth), was used for denaturing SDS-PAGE. Electrophoretic separation was conducted at room temperature for about 90-120 min at 140V, depending on the porosity of the gels employed. Gels intended for Western Blotting and post-translational modification analysis were not stained with Coomassie Brilliant Blue, but were immediately processed for further use as

117

Materials and Methods

described in the respective chapters. Unless stated otherwise, Precision Plus Unstained molecular weight standard from Bio-Rad was used.

6.2.6

Enzymatic Proteolysis

Following

separation

of

protein

mixtures

by

one-dimensional

polyacrylamide

gel

electrophoresis, the protein bands of interest were excised and in-gel digested. The gel bands were cut into cubes of about 1 mm3 and the Coomassie stain was removed by three consecutive washing steps employing 150 µl of a 50% (v/v) acetonitrile (ACN) / 50% (v/v) H2O solution. The gel pieces were incubated in the washing solution for 10 minutes on a shaker and the supernatants were discarded. In the next step, 80 µl ACN were added, the liquid was removed after five minutes, and was replaced by 80 µl of a 100 mM NH4HCO3 solution. Again, 80 µl ACN were added without removing the NH4HCO3 solution and the gel cubes were incubated for another ten minutes before the supernatant was discarded. In the following vacuum centrifugation step, the gel pieces were dried within 30 minutes. For reduction of disulfide bonds, 80 µl of a 10 mM dithiothreitol (DTT) / 100 mM NH4HCO3 solution were added and incubated at 56°C for 45 minutes. For alkylation of the reduced cysteines, the DTT solution was discarded and replaced by 80 µl of a 55mM iodoacetamide / 100 mM NH4HCO3 solution and incubated at ambient temperature in the dark for 30 minutes. For removal of the reagents, the gel pieces were washed by repeating the washing procedure described above and dried in the vacuum concentrator. Diverse proteases were used for different applications. Trypsin solution was prepared from 2µl-aliquots containing 1 µg trypsin in 1mM HCl. The aliquot was diluted with 18 µl of a 50 mM NH4HCO3 solution, resulting in a 50 ng/µl trypsin solution. Endoproteinase AspN solution was prepared by H2O to result in the desired concentrations (see respective descriptions). LysC, GluC, and chymotrypsin were used at 50 ng/µl. For enzymatic proteolysis, between 5 and 10 µl of enzyme solution were added to the dry gel pieces, depending on the total volume of the gel pieces and the original Coomassie staining intensity of the gel bands. After the protease solution was fully absorbed, the gel pieces were covered by a 50 mM NH4HCO3 solution. The gel pieces were incubated at 37°C for 16 hours unless stated otherwise. Upon termination of the digestion by adding 50 µl ACN / H2O / formic acid (47.5% (v/v) / 47.5 % (v/v) / 5% (v/v)) and retaining the supernatant, the peptides were extracted from the gel pieces by incubation at room temperature for 10 minutes with fresh solution for two more times. The individual supernatants were combined, concentrated in the vacuum concentrator to a volume of about 10-15 µl, and stored at –20°C for subsequent mass spectrometric analysis.

118

Materials and Methods

6.2.6.1

Enzymatic Proteolysis of Proteins for Peptide Mass Fingerprint Analysis

For characterization of amino acid sequences of CaM and melittin in-gel digestions were performed with trypsin (50 ng/µl, 37°C, over-night), trypsin / AspN mixture (50 ng/µl each, 37°C, over-night), LysC / AspN mixture (50 ng/µl each, 37°C, over-night), and AspN (40 ng/µl, 37°C, over-night), respectively. ANXA2 and p11 amino acid sequence coverages were obtained by analysis of digests with trypsin (50 ng/µl, 37°C, over-night) and AspN (30 or 60 ng/µl, 37°C, over-night). Chymotrypsin (50 ng/µl, 37°C, 2hrs) was additionally employed for in-gel digestion of p11 for obtaining better sequence coverage. In addition to trypsin, GluC (50 ng/µl, 37°C, 16 hrs) was used for characterization of the N-terminally truncated annexin A2 species. Co-purified proteins from A2t purification were digested with trypsin (50 ng/µl, 37°C, overnight), as were all the proteins of A2t binding partners (chapter 6.2.4). 6.2.6.2

Enzymatic Proteolysis of Calmodulin / Melittin Cross-Linking Reaction Mixtures

Trypsin alone (50 ng/µL) was used for all cross-linking reaction mixtures, whereas endoproteinase AspN (40 ng/µL), trypsin/AspN (both 100 ng/µl), and LysC/AspN (both 100 ng/µl) were used as additional digestion enzymes of the CaM / Mel complex cross-linked with EDC/sNHS. When employing a combination of two proteases, only half the volume of each was added to the gel bands to result in a two-fold dilution (i.e. 50 ng/µl) of the initial concentration. 6.2.6.3

Enzymatic Proteolysis of A2t Cross-Linking Reaction Mixtures

Trypsin (50 ng/µL) was used for all cross-linking reaction mixtures, whereas endoproteinase AspN (50 ng/µL) was exclusively used for the sDST cross-linking reaction mixtures. The digests were incubated at 37°C for 16 hrs.

6.2.7 MALDI-TOF Mass Spectrometry MALDI-TOFMS was performed on a Voyager DE™ RP Biospectrometry™ Workstation (Applied Biosystems) and on an Autoflex I instrument (Bruker Daltonik) both equipped with a nitrogen laser (337 nm). Both instruments possess delayed-extraction technology and were operated in positive ionization mode. Furthermore, both mass spectrometers possess a linear and a reflector detector. For analyses with the Voyager instrument 96-well stainless steel MALDI sample plates were used. An MTP 384 massive target T was the target plate for the Autoflex I instrument. Data acquisition and data processing were performed using the Voyager software version 5.1 and the Data Explorer software version 4.0 (Applied Biosystems), and the Flex Control 2.2.19.0 and Flex Analysis 2.2 software (Bruker Daltonik). 119

Materials and Methods

6.2.7.1

Sample Preparation for MALDI-TOFMS

Prior to MALDI-TOF mass spectrometric analysis the target plate onto which the samples are to be deposited needs to be thoroughly cleaned. This is achieved by rinsing with different solvents and gentle rubbing of the target surface. The samples were desalted prior to analysis employing either C-18 or C-4 ZipTips (Millipore) for low-volume peptide and protein samples, respectively. For larger volumes, microcon centrifugal filter devices (Millipore) with appropriate cut-off were employed. The matrices were prepared by preparing a saturated solution in 50-70 % (v/v) acetonitrile and 0.1 % (v/v) TFA or FA. The supernatant was several-fold diluted until a thinly and evenly dispersed matrix layer was observed upon deposition on the target. α-Cyano-4-hydroxy cinnamic acid (Bruker, (Sigma)) was employed for peptide analyses. Sinapinic acid (Sigma) was used for linear MALDITOFMS of the intact CaM/Mel complex. For linear MALDI-TOFMS of intact ANXA2 the following matrices were employed: 6-aza-2-thiothymine (ATT), 2’,5’-dihydroxyacetophenone , 2,5dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapinic acid), and 4hydroxyazobenzene-2-carboxylic acid (HABA). Matrix and sample solutions were deposited on the MALDI sample plate using the drieddroplet method. 6.2.7.2

Voyager DE RP

Linear mode MALDI-TOFMS of the non-digested CaM / Mel cross-linking reaction mixtures was performed for monitoring the extent of chemical cross-linking. Measurements were conducted in positive ionization mode using sinapinic acid as the matrix. The matrix was prepared in 30% (v/v) acetonitrile / 70% (v/v) H2O / and 0.1% (v/v) trifluoro acetic acid. Samples were desalted employing Microcon YM-10 filters (Millipore) and prepared using the dried droplet method. 100 shots were added to one spectrum in the mass range m/z 2000 to 42000. The instrument was calibrated using cytochrome c ([M+H]+average at m/z 12361.6) and myoglobin ([M+H]+average at m/z 16952.6). For sequence coverages of CaM, melittin, and ANXA2, the instrument was run in positive reflector mode with typically 200 shots summed to one spectrum. Calibration was performed with angiotensin II ([M+H]+mono at m/z 1046.54), angiotensin I ([M+H]+mono at m/z 1296.68), substance P ([M+H]+mono at m/z 1347.74), and somatostatin (red) ([M+H]+mono at m/z 1637.72). 6.2.7.3

Autoflex I

For protein analysis, measurements were performed in linear positive ionization mode using sinapinic

acid,

2,5-dihydroxybenzoic

acid

(DHB),

2’,5’-dihyroxyacetophenone,

4-

hydroxyazobenzene-2-carboxylic acid (HABA), and 6-aza-2-thiothymine (ATT) as matrices. 120

Materials and Methods

Positive ionization and reflectron mode were employed for MALDI-TOFMS measurements of peptide mixtures using α-cyano-4-hydroxy-cinnamic acid as matrix. All matrices were prepared in 50% (v/v) acetonitrile / 50% (v/v) H2O / and 0.1% (v/v) formic acid. Samples were desalted using C4-ZipTips for proteins and C18-ZipTips for peptides (Millipore), respectively, and prepared using the dried droplet method. For peptide analysis, 200 laser shots were added to one spectrum typically in the m/z ranges 800 to 4,000 or m/z 500 to 4,000. Peptide Calibration Standard (Bruker Daltonik) (angiotensin II ([M+H]+mono at m/z 1046.54), angiotensin I ([M+H]+mono at m/z 1296.68), substance P ([M+H]+mono at m/z 1347.74), bombesin ([M+H]+mono at m/z 1619.82), ACTH clip 117 ([M+H]+mono at m/z 2093.09), ACTH clip 18-39 ([M+H]+mono at m/z 2465.20), and somatostatin ([M+H]+mono at m/z 3147.47)) was used for external calibration of the mass spectra in the reflectron mode. Additionally, internal calibration of mass spectra was performed with autolytic peptide signals of trypsin. When analyzing AspN-digested samples of ANXA2 and p11, signals of ANXA2 and p11 peptides generated from AspN digestion that were frequently observed in the spectra, were used for internal calibration. For external mass spectra calibration in the linear mode, cytochrome c ([M+H]+average at m/z 12362), carbonic anhydrase ([M+H]+average at m/z 29025), and bovine serum albumin ([M+H]+average at m/z 66431) were used. 1000 laser shots were accumulated to one spectrum in the m/z range between 8,000 and 110,000. Ultraflex III: Experiments were conducted at Bruker Daltonik, Bremen.

6.2.8

Nano-High Performance Liquid Chromatography / Nano-Electrospray Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

6.2.8.1

Mass Spectrometric Analysis of the Calmodulin / Melittin Complex

The peptide mixtures from enzymatic digests were separated by nano-HPLC. Nano-HPLC was carried out on an Ultimate™ Nano-LC system (LC Packings) equipped with a Switchos II column switching module and a Famos™ Micro Autosampler with a 5-µl sample loop. Samples were injected by the autosampler and concentrated on a trapping column (PepMap, C18, 300 µm * 1 mm, 5 µm, 100 Å, LC Packings) with water containing 0.1% formic acid (v/v) at flow rates of 20 µl/min. After two minutes, the peptides were eluted onto the separation column (PepMap, C18, 75 µm * 150 mm, 3 µm, 100 Å, LC Packings), which had been equilibrated with 95% A (A being H2O + 0.1% (v/v) formic acid). Peptides were separated using the following gradient: 0-30 min: 5-50% B, 30-31 min: 50-95% B, 31-35 min: 95% B (B being acetonitrile + 0.1% (v/v) formic acid) at flow rates of 200 nl/min and detected by their UV absorptions at 214 and 280 nm.

121

Materials and Methods

The nano-HPLC system was coupled on-line to an Apex II FTICR mass spectrometer equipped with a 7 Tesla superconducting magnet (Bruker Daltonics) and a nano-electrospray ionization source (Agilent Technologies). For nano-ESIMS, coated fused-silica PicoTips (tip IDs 8 µm and 15 µm, New Objective) were applied. The capillary voltage was set to -1400 V. Mass spectral data were acquired over a range of m/z 400-2000, four scans were accumulated per spectrum, and 400 spectra were recorded for each LC/MS run. MS data acquisition was initialized with a trigger signal from the HPLC system 5 min after initiation of the LC gradient. Data were acquired over 34.5 min. Calibration of the instrument was performed with CID fragments (capillary exit voltage 200 V) of the LHRH peptide. Data acquisition and data processing were performed using the XMASS software, versions 5.0.10 and 6.0 (Bruker Daltonics). Processing of the raw data was performed using the ‘Projection’ tool in the XMASS software [Bruker BioAPEX User’s Manual, 1996]. 6.2.8.2

Mass Spectrometric Analysis of the ANXA2 / P11 Complex

The peptide mixtures from enzymatic digests were separated by the nano-HPLC system as described above. The only differences were that after three minutes peptides were eluted onto the separation column and that data acquisition and data processing were performed using XMASS version 7.02. 6.2.8.3

2

Tandem Mass Spectrometric Analysis of the ANXA2 / P11 Complex

In-gel digests of cross-linking reaction mixtures from heterodimeric and heterotetrameric complexes between ANXA2 and p11 were additionally analyzed by MS/MS experiments. The peptide mixtures resulting from the in-gel digests were separated by C18-RP-chromatography on a nano-HPLC system (Ultimate 3000, Dionex; pre-column: PepMap C18, 300µm * 5 mm, 3 µm, 100 Å, Dionex; separation column: PepMap, C18, 75 µm * 150 mm, 3 µm, 100 Å, Dionex; solvents A: 5% (v/v) acetonitrile, 0.1% (v/v) formic acid in water, B: 80% (v/v) acetonitrile, 0.08% (v/v) formic acid in water) using a gradient from 0 to 60 % B in 90 minutes followed by isocratic elution with 90% B for 3 minutes. An LTQ-FT mass spectrometer (Thermo Electron) with a 7 Tesla magnet equipped with a nano-ESI source (Proxeon Biosystems; emitter: distal coated PicoTips, tip i.d. 15 µm, New Objective) was on-line coupled to the nano-HPLC system. MS data were acquired over 100 min in data-dependent MS2 mode: each high-resolution full scan (m/z 300–2,000, resolution at m/z 400 was set to 100,000) in the ICR cell was followed by 10 product ion scans in the linear trap of the 10 most intense signals in the full-scan mass

2

Measurements on the ApexII instrument were conducted by Christian Ihling and Stefan Kalkhof.

122

Materials and Methods

spectrum (isolation window 3 u). Dynamic exclusion (exclusion duration 20 s, exclusion window ± 5 ppm) was enabled in order to allow detection of less abundant ions.

6.2.9

Processing of Mass Spectra

6.2.9.1

MALDI-TOFMS Data

3

Labeling of the mass spectra recorded on the Voyager (Applied Biosystems) instrument in the reflector mode was performed by the Deisotoping tool in the Data Explorer software (v. 4.0, Applied Biosystems). The obtained monoisotopic masses ([M+H]+) were manually evaluated by setting peak detection thresholds for defined detection ranges based on signal intensities. Linear MALDI-TOF mass spectra were labeled with the Peak Label tool in the Data Explorer software. Monoisotopic masses ([M+H]+) in the mass spectra recorded on the Autoflex I (Bruker Daltonik) in the reflector mode were labeled manually. Linear MALDI-TOF mass spectra were as well evaluated manually. 6.2.9.2

ESI-FTICRMS Data

ESI-FTICR (Apex II) mass spectra were processed with the XMass (v. 7.0.2 and 7.0.3, Bruker Daltonics) software. The single spectra were projected into one final mass spectrum using the Projection tool in the XMass software. The mass spectra were deconvoluted and monoisotopic masses ([M+H]+) were manually labeled. 6.2.9.3

MS/MS Data

Data analysis was performed by dividing the chromatograms of the total ion current into fiveminutes-fractions and the obtained average mass spectra were deconvoluted using the Mascot Distiller software v. 2.0 (Matrix Science). Product ion mass spectra of identified cross-linked peptides were also processed with Mascot Distiller (v. 2.0, Matrix Science).

6.2.10 Peptide Mass Fingerprint Analysis The Mascot (www.matrixscience.com) search tool Peptide Mass Fingerprinting was employed for the identification of unknown proteins obtained during A2t purification. A set of experimentally obtained peptide masses is compared to theoretical peptide masses calculated from protein sequences deposited in protein databases like e.g. the NCBI (National Center for Biotechnology Information, www.ncbi.nlm.nih.gov) database. Information on the taxonomy, the

3

Measurements on the LTQ-FT instrument were performed by Christoph Stingl.

123

Materials and Methods

employed protease, the number of missed cleavages, and on fixed (e.g. carbamidomethylation, N-terminal acetylation) and variable (e.g. methionine oxidation) modifications are provided by the user. Furthermore, the error window for experimental peptide masses needs to be defined. Then the entered MS peaklist is searched for matches and proteins putatively matching the entered masses are displayed. The probability-based MOWSE score given for an identified protein is a criterion whether the result is significant or not.

6.2.11

Data Analysis

Cross-linked products were identified using IsoFind, the GPMAW (General Protein Mass Analysis for Windows) software, versions 5.12beta3, 6.00, and 7.01 (Lighthouse Data) (available at: http//welcome.to/gpmaw), the ExPASy Proteomics tools in the Swiss-Prot Database (available at: www.expasy.org), ASAP (Automatic Spectrum Assignment Program) v. 1.09 and MS2Assign. 6.2.11.1 IsoFind The in-house developed tool ‘IsoFind’ (developed by Tibor Kohajda and Stefan Kalkhof) was employed for searching for distinct 4.025 or 8.05 amu spacing between two signals from a given MS peaklist. The macro is run in Excel and applied to MS peaklists containing m/z values in one column and either signal intensity or peak area values in the second column. From this, the macro additionally calculates the signal ratios based on either signal intensity or peak area. 6.2.11.2 General Protein Mass Analysis for Windows (GPMAW) This software calculates theoretical cross-linked products (inter- and intramolecular cross-linked products, peptides modified by a partially hydrolyzed cross-linking reagent) and peptides for one or two given protein sequences. Both the protease and cross-linking reagent employed are defined by the user as well as the maximum number of missed cleavages. The experimentally obtained masses from mass spectrometric analysis are compared to the theoretic values and are assigned to cross-linked products or peptides. Maximum mass deviations for correct assignment were 10 ppm for ESI-FTICRMS data and (at maximum) 100 ppm for MALDITOFMS data In case of doubt, cross-links assigned by the software were not considered as such when discrimination between a peptide and a cross-linked product or between two different crosslinked products, coincidently having the same mass, was ambiguous. In case of the CaM / melittin complex, cleavages by proteases at modified amino acids, such as the trimethylated K115 in CaM as well as amino acids modified by cross-linking reagents, were excluded. Both the N-terminus of CaM and the C-terminus of melittin were excluded from possible cross-linking 124

Materials and Methods

by EDC since the former is acetylated and the latter amidated. In case of the ANXA2 / p11 complex, cleavages by proteases at amino acids modified by cross-linking reagents, were allowed. The N-terminus of ANXA2 is acetylated and thus unavailable for cross-linking. 6.2.11.3 ExPASy Proteomics Tools The FindPept and FindMod tools of the Swiss-Prot database (www.expasy.org) were used for comparison of experimentally obtained m/z values from MS analysis of an enzymatically digested protein with the theoretical peptide masses calculated for a specified amino acid sequence. The FindPept tool was also used for searching for intrapeptide cross-links and peptides with a hydrolyzed cross-linker by defining the cross-linking reagent as variable modification. Data evaluation of the biotinylated protein was also performed using the FindPept and FindMod tools. The program also accounts for protease autolysis and contaminant keratin peptides. 6.2.11.4 Automated Spectrum Assignment Program (ASAP) The ASAP tool (http://roswell.ca.sandia.gov/~mmyoung/asap.html) was used for assigning m/z values obtained from MS analysis of enzymatically digested cross-linked proteins. ASAP assigns cross-linked products and peptides based on comparison to theoretical cross-linked products and peptides for a given protein. Information on cross-linker, protease, and amino acid modifications has to be provided. Unfortunately, ASAP works only for single proteins. Nevertheless, this program is useful as it, in contrast to GPMAW, considers variable modifications. 6.2.11.5 MS2 Assign MS2 Assign (http://roswell.ca.sandia.gov/~mmyoung/ms2assign.html) was used for assigning m/z values obtained from MS fragmentation data of cross-linked peptides. MS2Assign calculates a theoretical fragment library for a (cross-linked) peptide or a cross-linked pair of peptides. Peptide sequences, crosslinker (if applicable), and amino acid modifications have to be provided.

6.2.12

Computational Protein-Protein Docking

6.2.12.1 Protein-Peptide Docking of the CaM / Melittin Complex with Xplor-NIH Structures of the CaM / melittin complex were calculated by conjoined rigid body/torsion angle simulated annealing [Clore, 2000, Schwieters & Clore, 2001, Clore & Bewley, 2002] using the molecular structure determination package Xplor-NIH [Schwieters et al., 2003] (available on-line 125

Materials and Methods

at: http://nmr.cit.nih.gov/xplor-nih) and structures were viewed using the VMD-XPLOR visualization package [Schwieters & Clore, 2001] (available at: http://vmd-xplor.cit.nih.gov). Distance restraints derived from cross-linking data were represented by empirical -1/6 averages. This ensures that at least one of the distances in the restraint lies within the specified target range, but no penalty ensues if the remaining distances within the restraint are much longer than the target distance. The distances were classified into three ranges, ≤ 5 Å, ≤ 8.5 Å and ≤ 13.4 Å, corresponding to cross-links obtained using EDC, sDST and BS3, respectively. The starting coordinates employed for CaM and melittin were derived from the 2.2 Å resolution crystal structure of the CaM / smMLCK peptide complex (PDB accession code 1CDL [Meador et al., 1992) and the 2 Å resolution crystal structure of helical melittin (PDB accession code 2MLT [Eisenberg et al., 1980]), respectively. The N- (residues 5-70) and C-terminal (residues 81-146) halves of CaM, and the N- (residues 1-9) and C (residues 13-26) terminal halves of melittin were treated as rigid bodies. Residues 71-80 of calmodulin, residues 10-12 of melittin (i.e., the location of the kink between the two helical segments) and all interfacial side chains were given full torsional degrees of freedom. The non-bonded term in the target function comprised a quartic van-der-Waals repulsion term [Nilges et al., 1988] and a torsion angle database potential of mean force derived from high-resolution crystal structures [Clore & Kuszewski, 2002]. The latter ensures that the side chains torsion angles lie within energetically allowed rotamer ranges. A qualitative interpretation of the cross-linking data indicated that melittin could bind CaM in two opposing orientations. Consequently, both orientations were refined simultaneously by including two complete sets of non-overlapping coordinates (i.e., no interactions were allowed between the two sets).

4

6.2.12.2 Protein-Protein Docking of A2t with Rosetta The X-ray structures of N-terminally truncated human ANXA2 (PDB entry 1W7B [Rosengarth et al., 2004]) and the heterotetramer comprised of one p11 dimer (Homo sapiens) and two synthetic ANXA2 N-terminal peptides (PDB entry 1BT6 [Réty et al., 1999]) were used as starting structures for the docking procedure with Rosetta [Gray et al., 2003, Daily et al., 2005]. In a first low-resolution docking step 20,000 models were generated by randomizing both docking partners. The models were filtered using the four distance restraints listed in Table 4-5. The remaining 125 structures were clustered twice with cluster thresholds of 5 and 9 Å rmsd (root mean square deviation), respectively. The structures representing the centers of clusters with more than four models were used as starting structures for the following high-resolution docking run, allowing a perturbation of 5 and 9 Å, respectively. The resulting structures were filtered again (Table 4-5) and clustered with an rmsd threshold of 5 Å. The cluster center

4

Computational modeling of the calmodulin / melittin complex was performed by Dr. G. Marius Clore

126

Materials and Methods

structures of the top nine clusters with more than four models, together with two additional structures, retrieved from filtering for NZ-NZ distances of lysine side chains, were used for generating models of the symmetric A2t complex. A second ANXA2 monomer was added to the (p11)2 / ANXA2 complex by superimposition, utilizing the inherent symmetry of the p11 dimer. This superimposition, which was done with MAMMOTH [Olmea et al., 2002], led to steric clashes for three out of eleven structures. For the remaining eight structures, the missing eight amino acids between the N-terminal annexin peptide and N-terminally truncated ANXA2 as well as the four C-terminal amino acids of p11 were modeled based on secondary structure predictions using JUFO (www.meilerlab.org), PSIpred (http://bioinf.cs.ucl.ac.uk/psipred/), and SAM (http://www.cse.ucsc.edu/research/compbio/sam.html). Amino acid substitutions between porcine and human ANXA2 as well as between porcine ANXA2 and synthetic N-terminal ANXA2 peptide were conducted before the A2t structures were subjected to repacking and side-chain relaxation. With the objective of obtaining a model of the octameric ANXA2 / p11 complex, we created 2000 models of a p11 homotetramer by randomized docking. The starting structure for the subsequent dock perturbation run was chosen restricting the N- and C-termini of the two respective dimers to be in close proximity, thus ensuring that the opposite site that was found to be the ANXA2 binding site remained unoccupied. From the 2000 model structures after dock perturbation, one structure was picked, complying to the premise that the N- and Ctermini of the two respective dimers are equidistant and at the same time close to each other, thus obtaining a compact and symmetric homotetramer. The models of the heterooctamer structures between ANXA2 and p11 were obtained from the overlay of the A2t structure based on the geometry of the p11 homotetramer. The dimensions of the created octamer structures were determined using the Tabling function in the AMoRe software [Navaza, 1994]. 6.2.12.3

5

Determination of Solvent Accessibilities of Amino Acid Sidechains

Partially hydrolyzed cross-linkers, which are attached to lysine residues, provide valuable information on the solvent accessibilities of the respective amino acid side chains in a protein. The modified lysine residues were used for creating surface topology maps of ANXA2 and p11. The findings were validated by the GETAREA 1.1 software (www.scsb.utmb.edu/getarea, [Fraczkiewicz & Braun, 1998]) that calculates the solvent accessibilities of amino acid residues and assigns amino acid residues that display more than 50% solvent accessibility to be exposed on the surface of a protein.

5

The expert assistance of Stefan Kalkhof, who significantly contributed to computing the models of the complex between annexin

A2 and p11, is gratefully acknowledged.

127