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Cleavage of Fibrinogen Alpha Chains During Isoelectric Focusing Of J Electrophoresis 2007 ; 51 : 27 [Full Paper] Cleavage of fibrinogen alpha chains during isoelectric focusing of human plasma under non-denaturing conditions analyzed by micro two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry Takashi Manabe, Ya Jin, Nao Yamaguchi, Tetsuo Sugiyama and Kohei Ikari SUMMARY The identity of low-molecular-weight and minor protein spots, appeared in 2-DE pat- terns of human plasma, was examined. They were not obvious in the patterns of “Type-I” 2-DE (non-denaturing IEF followed by non-denaturing gel electrophoresis), but clearly detected in the patterns of “Type II” 2-DE (non-denaturing IEF followed by SDS gel elec- trophoresis) at pI 5.5–7.5 and apparent mass 8–40 kDa1). The spots were not obviously detected when the IEF gels were kept at low temperature (around 4°C) during electro- phoresis, suggesting that they are the proteolysis products of plasma proteins. The minor spots were more obviously detected when human plasma was subjected to ammonium sul- fate (AS) fractionation and the 0–35% saturated AS fraction was dialyzed and subjected to Type-II 2-DE. Then the 116 spots on the 2-DE pattern, detected at pI 5–7.5 and apparent mass 8–60 kDa, were excised and subjected to MALDI-MS measurements and the mass spectra were analyzed using the software of peptide mass fingerprinting (PMF) Mascot and ProFound to assign the proteins. Many of the spots were assigned to contain fibrinogen α chain, especially those at pI 5.5–7.5 and apparent mass 8–40 kDa, suggesting that these spots are its fragments. The distribution of the MS-detected peptide fragments suggested that the molecular-mass heterogeneity might be caused by the cleavage of multiple sites on the α chain. Care must be taken to keep the temperature of IEF gels at around 4°C dur- ing electrophoresis, when human plasma proteins are subjected to non-denaturing IEF. The absence of the spots of fibrinogen fragments on Type-II 2-DE gels would validate the intactness of plasma proteins. The advantages of micro gel system for the analysis of intact protein mixtures are suggested. Key words: two-dimensional electrophoresis, MALDI-MS, plasma protein, non-denaturing, fibrinogen alpha chain. trophoresis (Type-II 2-DE)2) and suggested that they might INTRODUCTION participate in blood coagulation1). Since the minor proteins Previously, we have reported a group of minor proteins could be enriched in 0–35% saturated ammonium sulfate in human plasma which were not detected when a plasma fraction of human plasma, the fraction was subjected to sample was subjected to non-denaturing isoelectric focus- Type-II 2-DE employing micro 2-DE system. The spots on ing (IEF) followed by non-denaturing pore-gradient gel a 2-DE gel at pI ca. 5–7.5 and apparent molecular mass ca. electrophoresis (Type-I 2-DE)2), but detected when it was 5–60 kDa were excised and subjected to matrix-assisted subjected to non-denaturing IEF followed by SDS gel elec- laser desorption-ionization mass spectrometry (MALDI- Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, Japan. Correspondence address: Takashi Manabe; Department of Chemistry, Faculty of Science, Ehime University, Matsuyama-City, Ehime 790-8577, Japan. Abbreviations: 2-DE, two-dimensional gel electrophoresis; AS, ammonium sulfate; IEF, isoelectric focusing; BPB, bromophenol blue; CBB, Coomassie brilliant blue R-250; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; PMF, peptide mass fingerprinting; PMSF, phenylmethylsulfonyl fluoride; IgG, Immunoglobulin G; IgM, Immunoglobulin M; FN, fibrinogen; α2M, α2-mac- roglobulin; Alb, albumin; PEG, polyethyleneglycol. (Received October 10, 2007, Accepted October 15, 2007, Published December 15, 2007) J Electrophoresis 2007 ; 51 : 28 MS) followed by the assignment of polypeptides with pep- run at 0.12 mA/tube constant current until a voltage of tide mass fingerprinting (PMF). 300 V was reached (about 30 min) and continued at 300 V/cm constant voltage for 40 min. The electrode solutions for MATERIALS AND METHODS IEF (0.01 M H3PO4 for anode and 0.04 M NaOH for cath- 1. Materials ode) were cooled to 4°C before electrophoresis and the Ammonium persulfate, Bis, N,N,N',N'-tetramethylethyl- anode solution, in which the IEF gel columns were dipped, enediamine, all special grade for electrophoresis, and was kept around 4°C using an ice bath. In some cases, the triflouroacetic acid (TFA) (25% aqueous solution), grade for temperature of the electrode solutions was changed to protein structure analysis, were from Wako Pure Chemical examine the effects on the protein separation patterns. The Industries (Osaka, Japan). Acrylamide, special grade for IEF gels were transferred onto the polyacrylamide micro electrophoresis, was from Daiichi Pure Chemicals Co. slab gels (8.4%–17.85% T linear gradient, 5% C, (Tokyo, Japan). Ampholines pH 3.5–10 and pH 3.5–5 were 38×38×1 mm, containing 1% SDS), equilibrated for 10 min from Amersham Biosciences AB (Uppsala, Sweden). in a 0.01 M Tris–0.076 M glycine buffer–2% SDS (pH 8.3), Coomassie brilliant blue R-250 (CBB) was from Fluka and electrophoresis was run at 10 mA/gel constant current Chemie AG (Buchs, Switzerland) and porcine sequencing- with an electrode buffer of 0.05 M Tris–0.38 M glycine– grade modified trypsin was from Promega (Madison, WI, 0.1% SDS (pH 8.3, containing 1 g/L bromophenol blue). USA). α-Cyano-4-hydroxycinnamic acid (4-CHCA), human The electrode buffer solution was 0.1% SDS–0.05 M gly- adrenocorticotropic hormone fragment (ACTH 18–39) for cine–0.38 M Tris (pH 8.3). Electrophoresis was continued internal calibration of MALDI-MS, and phenylmethylsulfo- until the band of BPB (added in the cathode buffer solution) nyl fluoride (PMSF) were from Sigma (St. Louis, MO, moved to about 3 mm from the bottom end of the slab gels USA). ZipTipµC18 was from Millipore (Bedford, MA, USA). (about 50 min). The micro slab gels were stained in 0.1% Water for all the solutions was prepared with a Milli-Q CBB in 50% methanol/7% acetic acid (v/v) for 15 min and Gradient A-10 water system (Millipore). Human plasma destained in 20% methanol/7% acetic acid (v/v) for 150 min was from an apparently healthy individual (28 y, female) (one change). The gels were rehydrated in 7% v/v acetic preventing the blood clotting with 0.045% heparin, supple- acid and stored in 7% v/v acetic acid at 4°C until use. Silver mented with sucrose to a final concentration of 40% w/v, staining was done according to Oakley, et al.5) with some and stored in small aliquots at –20°C. modifications for micro slab gels. 2. Ammonium sulfate fractionation of plasma sample 4. In-gel digestion of the protein spots Small portions of ammonium sulfate (AS) powder were The stained spots on the Type-II micro 2-DE gel were added to the plasma sample (1 mL) and dissolved until the subjected to the procedures of in-gel digestion with trypsin concentration of AS reaches at 35% saturation at 4°C. The as described in detail6), except the steps of reduction and solution was centrifuged at 14,000×g for 10 min and the alkylation were not employed. precipitate was dissolved with a minimum volume of water. The solution was dialyzed against 0.0375% heparin solution 5. MALDI-TOF MS of tryptic peptides and peptide overnight at 4°C, supplemented with 20% sucrose, and cen- mass fingerprinting trifuged at 14,000×g for 10 min. The supernatant solution Mass spectra of tryptic peptides were acquired with was designated as 0–35% AS fraction. In some cases, the a Voyager-DE PRO MALDI-TOF mass spectrometer AS precipitate was dissolved with a 0.05% heparin solution (Applied Biosystems) operated in reflector mode at 20 kV and subjected to dialysis. accelerating voltage and 100 nsec ion extraction delay with a nitrogen laser working at 337 nm and 3 Hz in m/z range 3. Micro 2-DE 600–4000. Monoisotopic peaks of trypsin autodigest (m/z An apparatus for micro 2-DE3) and an automated appara- 842.51 and 2211.10) were used for internal calibration. The tus for gradient gel preparation4) have been used. Type-II 2- program Data Explorer (Applied Biosystems) was used to DE was performed as described previously2) with some process the data and to prepare mass peak lists using the modifications as described below. The plasma sample following functions of the software; 1) Baseline Correction, (1 µL) or the 0–35% AS fraction (8 µL) were subjected to 2) Advanced Baseline Correction, 3) Noise Filter/ isoelectric focusing (IEF) in the absence of denaturants Smoothing, 4) Manual Mass Calibration, 5) Peak Detection, employing polyacrylamide column gels (4.2% T, 5% C, in the m/z ranges of 750–1000, 1000–1500, 1500–2000, 1.4 mm φ×35 mm) which contained Ampholine pH 3.5–10 2000–2500, 2500–4000 with the thresholds of peak height with a final concentration of 2%. Ampholine pH 3.5–5, and peak area being manually decided in each range, 6) which we further added with a final concentration of 0.5% in Filter Peak List, set as monoisotopic and charge state 1, our routine protocol of non-denaturing IEF2), was omitted and 7) Peak Deisotoping, set as proton adduct and generic in order to improve the resolution around pI 5–8. IEF was formula C6H5NO. PMF programs of Mascot and ProFound J Electrophoresis 2007 ; 51 : 29 (Ver. 4.10.5) were used with the following parameters: pI 5.5–7.5 and mass 8–45 kDa1) are not obvious in the 2-DE Database, Swiss-Prot ver.
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