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Using low-molecular mass pI markers in proteomic staining-free method for study of posttranslationally modified proteins Karel Mazanec 1,2 , Karel Šlais 2 and Josef Chmelík 2 1 Institute of Material Chemistry, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech Republic, e-mail: mazanec@iach.cz 2 Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veveří 97, 602 00 Brno, Czech Republic INTRODUCTION At present the knowledge of proteome plays very important role in the study of living processes. Special attention in our lab is paid to the investigation of the proteome of barely grains. The aim is to contribute to understanding of malting processes and to selection of malting barley cultivars. Proteins are the major functional molecules of life made of amino acids arranged in a linear chain linked together by peptide bonds. The residues in a protein are often chemically altered in a process known as post-transcriptional modification (PTM): either before the protein can function in the cell, or as part of control mechanisms. The identification of the PTM is experimentally difficult. Due to the fact that many samples, mainly of biological origin, are complex mixtures of proteins with a wide range of molecular masses, salts and other compounds, proteins should be separated and purified prior to their identification by mass spectrometry. Traditionally, most proteomics researches are based upon two-dimensional gel electrophoresis involving isoelectric focusing (IEF) as one dimension. Separated compounds are focused into very sharp bends according to their pI values during IEF. They are twice or more concentrated in this zones. However, ampholytes present in gel and creating pH gradient during IEF complicate the visualizing the separated proteins by staining and make this method impracticable for common proteomic utilization. The aim of this work was to develop and optimize the novel staining-free proteomic procedure for separation of intact proteins by gel IEF in presence of low-molecular mass pI markers and subsequent determination of molecular masses of separated compounds in the gels by MALDI-TOF/TOF MS. The selected set of pI markers includes both colored and colorless compounds with known low-molecular mass. Thus, the identification of both pI marker and proteins in the same excised piece of gel by MS technique is expected to give reliable information about the correct pI values of analyzed proteins even in complex samples. MATERIALS AND METHODS pI markers – The substituted phenols (I - Mw 314.08, pI 3.9, yellow color; II - Mw 359.10, pI 4.3, orange; III - Mw 272.06, pI 5.3, yellow; IV - Mw 252.12, pI 5.7, colorless; V - Mw 285.09, pI 6.4, yellow; VI - Mw 315.10, pI 7.0, yellow; VII - Mw 337.17, pI 7.5, yellow; VIII – Mw 265.14, pI 7.9, yellow; IX - Mw 363.23, pI 8.4, yellow; X - Mw 267.16, pI 8.9, yellow; XI - Mw 352.20, pI 9.0, colorless; XII - Mw 333.21, pI 10.1, yellow) used as pI markers were prepared by the procedure described by Šlais and Friedl 1 at the Institute of Analytical Chemistry (Brno, Czech Republic). The pI values were determined by potentiometric titration. The chemical structures of pI markers are shown in Fig. 1. Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006 Sekce DSP 2006, strana 254
Fig.1: Chemical structures of pI markers used in this work. Polyacrylamide gels and IEF - The protein samples (2 mg/ml) were mixed with pI markers (10 mg/ml each) and the 4 μl of the mixtures were loaded onto the polyacrylamide gel (16%). The carrier BioLyte 3/10 ampholyte (final concentration 2%) (Bio-Rad, CA, USA) were added prior to gel polymerization. Separation was performed with constant electric power of 0.6 W for 2 h supplied by VNZ 22 power supply (CSAV Development Workshop, Czech Republic) using a model 111 Mini IEF Cell (Bio-Rad, CA, USA). After focusing completion the gels were gently removed from electrodes and scanned. Ladders of color pI markers simplify the orientation in the gel even when the separated proteins cannot be seen. Thus, the bands with proteins were excised according to positions of pI markers. The excised gel pieces were then transferred to 0.5 ml tubes. The pI markers were then eluted from gel by 30 μl water/ethanol 1:1 (v/v) for 15 min. Protein digestion and identification - Excised gel pieces after the extraction of the pI markers were washed twice with water/acetonitrile 1:1 (v/v) for 15 min. Then proteins were identified according to standard proteomic protocol utilizing in-gel digestion of reduced and alkylated proteins with trypsin. 2 For comparison, the other IEF focused gels were stained with Coomassie Brilliant Blue R 250. Due to the carrier 3/10 ampholytes used for creating of pH gradient in gels precipitated the Coomassie dye during staining causing strong blue haze on the background, the washing step (10% TCA in water/methanol (10/3 v/v); 12-14 h) had to be inserted prior to own staining procedure. Mass spectrometry - MS measurements were carried with 4700 Proteomics Analyzer (Applied Biosystems, USA) MALDI TOF/TOF mass spectrometer (equipped with Nd/YAG laser; 355 nm). Argon was used as a collision gas. A solution of sinapinic acid (SA) (20 mg/ml in acetonitrile/water (3/2 v/v)) was used as matrix for markers. Alpha-cyano-4hydroxycinnamic acid (CHCA) (both Sigma-Aldrich, Germany) was used as matrix for peptides in concentration of 10 mg/ml acetonitrile/water (3/2 v/v). Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006 Sekce DSP 2006, strana 255
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PRODUCTION OF SELECTED SECONDARY ME
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containing ampicillin, IPTG and x-g
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THE SIMPLE METHOD FOR THE RECOGNITI
Page 7 and 8: Figure 1 Negative-ion mode MALDI-TO
Page 9 and 10: Table 1 Evaluation of negative-ion
Page 11 and 12: Fig. 1: Scheme of the hydride gener
Page 13 and 14: [4] H. Matusiewicz, M. Kopras, J. A
Page 15 and 16: - are hypotriglyceridemic; - exhibi
Page 17 and 18: HYDROPHOBIZED SODIUM HYALURONATE IN
Page 19 and 20: spectrophotometer (AMINCO-Bowman, S
Page 21 and 22: Hyaluronate derivatives showed with
Page 23 and 24: copolymers were additionally functi
Page 25 and 26: Each micelle has a hydrophobic core
Page 27 and 28: STUDY OF THE COPPER(II) IONS NON-ST
Page 29 and 30: CuSO4, Cu(ClO4)2 and Cu2P2O7 of the
Page 31 and 32: total flux [mol.m -2 ] 0.7 0.65 0.6
Page 33 and 34: number of free radicals is very low
Page 35 and 36: Differences can be caused by severa
Page 37 and 38: MEASUREMENT OF THERMOPHYSICAL PROPE
Page 39 and 40: The typical time responses of tempe
Page 41 and 42: Figure 4 illustrates the typical de
Page 43 and 44: sodium sulphate and filtered to a 5
Page 45 and 46: Compare all tested evening primrose
Page 47 and 48: IMPROVED FLUORIMETRIC DETERMINATION
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Page 51 and 52: F i 60 50 40 30 20 10 Data UCL LCL
Page 53 and 54: PHYSICAL-CHEMICAL ASPECTS OF PAINT
Page 55 and 56: Finally, the solubility parameter o
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Page 61 and 62: Identification of pI markers - Firs
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