J Electrophoresis 2007 ; 51 : 27

[Full Paper]

Cleavage of 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 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 . 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. 41.11, taxonomy, homo sapiens; pattern, but when the gel was further stained with silver mass tolerance, 50 ppm; maximum 1 missed cleavage per (Fig. 1B), more than 50 spots were detected in the corre- peptide, partial oxidation at methionine residues. sponding region. When the plasma sample was added 1 mM PMSF, kept on ice bath for 15 min, and subjected to the IEF RESULTS AND DISCUSSION run at 30°C, the minor spots still appeared in the silver- 1. Effects of temperature during IEF analyzed by stained 2-DE pattern (Fig. 1C). However, when the IEF Type-II 2-DE was run with electrode solutions kept around 4°C, almost Figure 1 shows micro 2-DE patterns of a human plasma all the minor spots were not detected on the silver-stained sample (1 µL) using non-denaturing IEF in the first dimen- 2-DE pattern (Fig. 1D). IEF run at 25°C also showed a sion and SDS gel electrophoresis (8.4%–17.85% T and 5% silver-stained 2-DE pattern with the minor spots, but with C polyacrylamide gradient gel) in the second dimension lesser densities of the spots compared with the 30°C pat- (Type-II 2-DE)2). When IEF was run with electrode solu- tern (data not shown). These results strongly suggest that tions kept around 30°C and the 2-DE gel was stained with the minor spots represent the proteolysis products of CBB, human plasma proteins were separated as shown in human plasma proteins. Also, it is suggested that the addi- Figure 1A. The minor spots which had been detected at tion of protease inhibitors might not be effective to prevent

Fig. 1. The effects of the temperature of electrode solutions during IEF. A): A plasma sample (1 µL) was subjected to non-denaturing IEF keep- ing the temperature of the IEF electrode solutions at 30°C and then the Type-II micro 2-DE pattern was obtained by staining the gel with CBB. B): The gel shown in A) was further subjected to silver stain- ing. C): The plasma sample was supplemented with 1 mM PMSF (final concentration) and the same protein quantity as in A) (1.25 µL of the mixture) was subjected to IEF at 30°C and the micro 2-DE gel was CBB stained, and then silver stained. D): The plasma sample (the same as in A, 1 µL) was subjected to non-denaturing IEF keep- ing the temperature of the IEF electrode solutions at 4°C and then the micro 2-DE gel was CBB stained, and then silver stained. The locations of albumin (Alb) and IgG are indicated. J Electrophoresis 2007 ; 51 : 30 the proteolysis compared with the temperature control. The previous results that the minor spots have been detected on the large 2-DE gel by CBB staining (IEF gel 160 mm long and slab gel 160 mm×120 mm)1) might be explained by the long IEF time, 2 mA contant current for 1 h and 510 V constant voltage for 20 h at 7°C. In the case of micro 2-DE, we employ much shorter IEF time (0.12 mA constant current for ca. 30 min and 300 V constant voltage for 40 min at 4°C) and this might explain why the minor spots were not obvious on the CBB patterns of micro 2-DE gels, even after the IEF run at 30°C (Fig. 1A). We have also reported the minor spots on a micro 2-DE gel, when the plasma sample volume was increased to 4-fold (4 µL) and the gel was subjected to silver staining1). Since we could not detect the spots in the range of pI 5.5–7.5 and mass 8– 45 kDa when IEF was run at 4°C (Fig. 1D), we conclude that our temperature control during micro IEF in the previ- ous paper was insufficient. Care must be taken to keep the temperature at around 4°C during electrophoresis when Fig. 2. A Type-II micro 2-DE pattern of the 0–35% AS fraction complex protein mixtures are separated in the absence of of a human plasma sample. denaturing agents, in order to minimize the possibility of A human plasma sample was subjected to ammonium sulfate proteolysis. fractionation and the 0–35% AS fraction was dissolved in water and dialyzed against a 0.0375% heparin solution. The dialyzed solution (20 µL, ca. 400 µg protein) was subjected to 2. Type-II 2-DE of 0-35% AS fraction and MALDI- non-denaturing IEF keeping the temperature of the IEF TOF MS-PMF analysis of the stained spots electrode solutions at 4°C and then a Type-II micro 2-DE Since we found that the minor proteins are produced pattern was obtained by staining the gel with CBB. The spots in the region of pI 5–8 and mass 8–60 kDa were num- during IEF in the absence of denaturing agents, we aimed bered, excised, and subjected to in-gel digestion, MALDI- to assign the proteins using MALDI-TOF MS and PMF. TOF MS, and peptide mass fingerprinting. The locations of α α However, in our protocol of MALDI-MS and PMF we albumin, IgG, 2-macroglobulin ( 2M), and fibrinogen (FN) are indicated. needed a protein spot which is detectable by CBB staining for reliable assignment of its constituent polypeptide6). Therefore, we expected that the minor spots on the 2-DE would keep working during dialysis. However, when we gels shown in Fig. 1B, which were obvious only after silver dissolved the 0–35% AS precipitate with 0.05% heparin and staining, would not be suited for the analysis. Previously, dialyzed against 0.0375% heparin solution, the Type-II 2- we have reported that the minor proteins are likely to form DE pattern of the dialyzate showed much less quantities of high-molecular-mass proteins under non-denaturing condi- the minor proteins (data not shown). These results sug- tions and they are present in 0–4% PEG fractions or in 0– gested that the minor proteins would represent the prod- 35% AS fractions1). Then we employed AS fractionation to ucts of proteolysis in the steps of blood enrich them and the 0–35% AS fraction (described in Sec- reactions. Blood clotting is known to be inhibited by anti- tion 2.2, ca. 400 µg protein) was subjected to Type-II micro III in the presence of heparin7), but the separation 2-DE keeping the temperature of the IEF electrode of III from the mixtures of proteases and their solutions at 4°C. As shown in Fig. 2, more than 100 CBB- substrate proteins during IEF might initialize the proteoly- stained spots were detected in the region of pI 5–8 and sis. Similar situation might happen when the concentration mass 8–60 kDa and their locations are quite similar with of heparin was lowered by dissolving the 0–35% AS precip- those of the minor spots detected in the 2-DE pattern of the itate with water. According to these considerations, we plasma sample (Fig. 1B). These results suggest that the decided to assign the spots in Fig. 2, in the region of pI 5–8 processes of 0–35% AS fractionation included step(s) of and mass 8–60 kDa, in order to confirm the assumptions. proteolysis of plasma proteins. Then we re-examined each process of AS fractionation and concluded that the step of 3. Assignment of the stained spots on the Type-II 2- dialysis of the 0–35% AS precipitate against 0.0375% hep- DE gel of 0–35% AS fraction by MALDI-MS and PMF arin solution is the most plausible step which might cause The numbered spots in Fig. 2 were excised and the the proteolysis. We have dissolved the precipitate with proteins in each gel piece was subjected to in-gel digestion, water then the solution was subjected to dialysis, assuming the peptide fragments were extracted, concentrated with that the heparin added in the step of plasma preparation ZipTipµC18, and subjected to MALDI-MS analysis as J Electrophoresis 2007 ; 51 : 31

Table 1. Identification of protein spots by MALDI-TOF MS and two programs of PMF Mascot Results ProFound Results spot 1st protein cover- 1st probability cover- No. protein namea) protein nameb) Candidate score age (%)c) candidate (Z value) age (%)d) 1 P25311 zinc-zlpha-2-glycoprotein 131 44 P25311 zinc-zlpha-2-glycoprotein 2.38 45 2 P25311 zinc-zlpha-2-glycoprotein 147 47 P25311 zinc-zlpha-2-glycoprotein 2.35 47 3 P06727 apolipoprotein A-IV 241 60 P06727 apolipoprotein A-IV 2.40 57 4 P06727 apolipoprotein A-IV 58 21 P04264 cytokeratin 1 1.14 19 5 e) P26927 macrophage stimulating protein 1.87 12 6 e) f) 7 e) f) 8 P04264 cytokeratin 1 72 20 P04264 cytokeratin 1 1.94 19 9 P04264 cytokeratin 1 54 17 P04264 cytokeratin 1 1.34 15 10 Q14624 35kDa ITI heavy chain H4 51 15 P04264 cytokeratin 1 1.25 16 11 e) P04264 cytokeratin 1 2.21 20 12 P01024 complement C3 85 11 P01024 complement C3 2.32 10 13 P01024 complement C3 90 11 P01024 complement C3 2.38 10 14 Q14624 35kDa ITI heavy chain H4 67 13 Q14624 35kDa ITI heavy chain H4 2.23 14 15 Q14624 35kDa ITI heavy chain H4 78 14 Q14624 35kDa ITI heavy chain H4 2.37 15 16 e) f) 17 P02671 fibrinogen alpha chain 67 14 P02671 fibrinogen alpha chain 2.20 11 18 Q14624 35kDa ITI heavy chain H4 63 13 Q14624 35kDa ITI heavy chain H4 2.36 13 19 e) f) 20 P02671 fibrinogen alpha chain 58 18 P02671 fibrinogen alpha chain 1.85 13 21 P02671 fibrinogen alpha chain 58 18 P02671 fibrinogen alpha chain 2.23 14 22 P02671 fibrinogen alpha chain 64 17 P02671 fibrinogen alpha chain 2.39 13 23 P02671 fibrinogen alpha chain 82 19 P02671 fibrinogen alpha chain 2.20 14 24 e) P02671 fibrinogen alpha chain 1.72 11 25 P02671 fibrinogen alpha chain 107 26 P02671 fibrinogen alpha chain 2.29 18 26 P02671 fibrinogen alpha chain 58 17 P02671 fibrinogen alpha chain 2.36 13 27 e) P02671 fibrinogen alpha chain 1.17 10 28 e) f) 29 e) f) 30 e) f) 31 e) f) 32 P02671 fibrinogen alpha chain 65 17 P02671 fibrinogen alpha chain 2.34 13 33 P02671 fibrinogen alpha chain 70 20 P02671 fibrinogen alpha chain 2.18 14 34 P02671 fibrinogen alpha chain 77 19 P02671 fibrinogen alpha chain 2.27 14 35 P02671 fibrinogen alpha chain 77 19 P02671 fibrinogen alpha chain 2.14 14 36 P02671 fibrinogen alpha chain 116 22 P02671 fibrinogen alpha chain 2.28 17 37 e) P02671 fibrinogen alpha chain 2.06 13 38 e) f) 39 e) P35527 keratin CK9 1.13 12 40 P02671 fibrinogen alpha chain 68 16 P02671 fibrinogen alpha chain 2.18 12 41 P02671 fibrinogen alpha chain 78 19 P02671 fibrinogen alpha chain 2.21 14 42 P02671 fibrinogen alpha chain 76 20 P02671 fibrinogen alpha chain 2.23 15 43 P02671 fibrinogen alpha chain 68 18 P02671 fibrinogen alpha chain 1.96 14 P02671 44 P35527 Keratin, type I cytoskeletal 9 54 22 fibrinogen alpha chain+cytokeratin 1 1.17 12 19 P35527 45 P04264 cytokeratin 1 54 16 P04264 cytokeratin 1 2.26 17 P02671 fibrinogen alpha 72 18 P02671 fibrinogen alpha 46 2.16 19 14 P04264 chain+cytokeratin 1 69 19 P04264 chain+cytokeratin 1 47 e) f) 48 e) P04264 cytokeratin 1 1.65 15 49 P02647 apolipoprotein A-1 87 33 P02647 apolipoprotein A-1 2.40 70 50 P02647 apolipoprotein A-1 173 59 P02647 apolipoprotein A-1 2.31 59 51 e) f) 52 e) f) 53 P02671 fibrinogen alpha chain 66 21 P02671 fibrinogen alpha chain 2.25 20 54 e) P02671 fibrinogen alpha chain 2.25 11 55 e) f) 56 e) P02671 fibrinogen alpha chain 1.62 9 57 e) f) 58 e) P02671 fibrinogen alpha chain 2.07 9 J Electrophoresis 2007 ; 51 : 32

59 e) P02671 fibrinogen alpha chain 1.62 8 60 P02671 fibrinogen alpha chain 76 19 P02671 fibrinogen alpha chain 2.36 14 61 e) f) 62 e) f) 63 P02753 plasma retinol binding protein 66 47 P02753 plasma retinol binding protein 2.31 48 64 P02753 plasma retinol binding protein 96 60 P02753 plasma retinol binding protein 2.17 61 65 e) P02671 fibrinogen alpha chain 2.17 13 66 P02671 fibrinogen alpha chain 108 21 P02671 fibrinogen alpha chain 2.18 16 67 P22352 plasma glutathione peroxidase 52 25 P22352 plasma glutathione peroxidase 1.40 23 68 P22352 plasma glutathione peroxidase 82 38 P22352 plasma glutathione peroxidase 2.03 35 69 e) f) 70 e) P34896 serine methylase 1.00 71 P02671 fibrinogen alpha chain 70 19 P02671 fibrinogen alpha chain 2.37 15 72 P02671 fibrinogen alpha chain 86 22 P02671 fibrinogen alpha chain 2.40 17 73 P02671 fibrinogen alpha chain 59 17 P02671 fibrinogen alpha chain 2.37 13 74 e) f) 75 P02671 fibrinogen alpha chain 81 19 P02671 fibrinogen alpha chain 2.31 14 76 P02671 fibrinogen alpha chain 72 19 P02671 fibrinogen alpha chain 2.25 15 77 P02671 fibrinogen alpha chain 85 20 P02671 fibrinogen alpha chain 2.33 15 78 P02671 fibrinogen alpha chain 65 18 P02671 fibrinogen alpha chain 2.40 16 79 P02671 fibrinogen alpha chain 56 11 P02671 fibrinogen alpha chaing) 2.06 8 80 P02671 fibrinogen alpha chain 52 9 P02671 fibrinogen alpha chain 1.90 7 81 P02671 fibrinogen alpha chain 83 18 P02671 fibrinogen alpha chaing) 2.28 16 82 P02671 fibrinogen alpha chain 63 11 P02671 fibrinogen alpha chaing) 2.05 14 83 P02671 fibrinogen alpha chain 76 14 P02671 fibrinogen alpha chain 1.92 10 84 e) f) 85 e) P02671 fibrinogen alpha chain 0.97 6 86 e) P02671 fibrinogen alpha chain 1.45 10 87 P02671 fibrinogen alpha chain 51 11 P02671 fibrinogen alpha chain 2.27 8 88 e) f) 89 e) P02671 fibrinogen alpha chain 0.44 6 90 P02671 fibrinogen alpha chain 60 14 P02671 fibrinogen alpha chain 2.07 15 91 P08686 cytochrome P450 XXIB 52 12 P02671 fibrinogen alpha chain 1.43 11 92 e) P02671 fibrinogen alpha chain 0.97 7 93 P02671 fibrinogen alpha chain 50 9 P02671 fibrinogen alpha chain 1.44 10 94 e) P02671 fibrinogen alpha chain 1.92 10 95 e) P02671 fibrinogen alpha chaing) 1.52 12 96 P02671 fibrinogen alpha chain 61 18 P02671 fibrinogen alpha chaing) 2.14 14 97 P04264 cytokeratin 1 53 14 P04264 cytokeratin 1 2.04 14 98 e) P02671 fibrinogen alpha chain 0.96 9 99 P02671 fibrinogen alpha chain 50 7 P02671 fibrinogen alpha chain 0.96 6 100 e) fibrinogen alpha chain 0.99 6 101 e) f) 102 e) f) 103 e) f) 104 e) P02671 fibrinogen alpha chain 1.23 10 105 O14511 pro-neuregulin-2 54 9 P02671 fibrinogen alpha chain 0.87 10 106 P02671 fibrinogen alpha chain 56 11 P02671 fibrinogen alpha chain 1.83 8 107 P02671 fibrinogen alpha chain 54 8 P02671 fibrinogen alpha chain 1.22 6 108 e) f) 109 e) P02671 fibrinogen alpha chain 1.13 110 e) P02671 fibrinogen alpha chain 1.09 6 111 e) f) 112 e) f) 113 e) f) 114 e) f) 115 P04264 cytokeratin 1 63 21 P04264 cytokeratin 1 2.10 16 116 e) P04264 cytokeratin 1 1.17 10 a) Proteins with scores higher than 63 are shown in bold. b) Proteins with Z values larger than 1.69 are shown in bold. c) Fibrinogen alpha chain coverage was calculated from sequence 1–644. d) In some cases, fibrinogen alpha chain coverage was calculated from sequence 1-866. e) No candidate with score higher than 49. f) No candidate with Z value higher than 0.8. g) Mass tolerance was set at 500 ppm. J Electrophoresis 2007 ; 51 : 33 described in Sections 2.4 and 2.5. The results are summa- single polypeptide, fibrinogen alpha chain, we examined the rized in Table 1. In the case of Mascot search, out of the 100 results of PMF assignment and compared the location of spots from spot number 17–116, 26 spots were assigned to the assigned peptides in the amino acid sequence of fibrino- be fibrinogen alpha chain with protein scores larger than 63 gen alpha chain. Table 2 shows the list of fibrinogen pep- (p<0.05) and another 13 spots were suggested to be - tides detected in the spots which have been assigned to be ogen alpha chain with protein scores lager than 49. In the fibrinogen at high confidence level. As shown in the Table, case of ProFound (Ver. 4.10.5) search, 43 spots out of the all the spots (except Spot 25) lacked the C-terminal pep- 100 were assigned to be fibrinogen alpha chain with Z-value tides from G592. The spots with relatively high apparent larger than 1.69 (p<0.05) and another 18 spots were sug- mass (Spot 17 to Spot 77) provided the mass peaks of the gested to be fibrinogen alpha chain with Z-value lager than peptide L250-R258 or M259-R271 and then followed the 0.87. (Recently, the algorithm of ProFound has been peptides up to the C-terminal region R591. On the other changed and we are not sure the same results will be hand, the spots with low apparent mass (Spot 82 to Spot obtained form the current version.) Most of the spots 106) lacked the N-terminal peptides up to V449. These which have been assigned to be fibrinogen alpha chain results suggest that the minor spots might be produced by reside at pI 5.5–7.5 and apparent mass 8–40 kDa. These the cleavage of fibrinogen α-chains at L250-R258 or M259- results strongly suggest that the minor spots detected on R271, at the C-terminal sides of six lysine or arginine resi- Type-II 2-DE patterns of human plasma with long IEF time dues in the region of K432-K448, and at the C-terminal or at elevated temperature would represent the fragments sides of R591 and/or K599, and then further cleavage of fibrinogen alpha chain, since intact alpha chain has molec- followed to produce the multiple spots. Pizzo, et al.7) exam- ular mass of 91 kDa (isoform 1) or 66 kDa (isoform 2). ined the digestion of fibrinogen by and found that at the early stages, fibrinogen α-chains were extensively 4. Distribution of the PMF-assigned peptides in the degraded producing heterogeneous products, whereas β amino acid sequence of fibrinogen alpha chain and γ chains were left intact. Takagi, et al. 8) deduced the In order to understand the origin of multiple spots from a early plasmin attacking points by determining the amino

Table 2. Peptides assigned to be the fragments of fibrinogen alpha chain by PFM in±25 ppm tolerance peptide asignment represented by amino acid sequence in fibrinogen alpha chain spot number 70– 98– 244– 250– 259– 272– 288– 426– 449– 468– 481– 511– 528– 548– 559– 582– 600– 84 114 249 258 271 287 308 432 458 476 510 527 547 558 573 591 621 17 22 23 25 32 33 34 35 36 40 41 42 43 46 53 60 66 71 72 75 76 77 78 81 82 83 90 96 106 J Electrophoresis 2007 ; 51 : 34 acid sequence of the plasmin-derived peptide. Four plasmin immunoglobulin G-associated minor proteins in human attacking points in fibrinogen α-chains were proposed; plasma by nondenaturing and denaturing two-dimensional K225-M226, K238-S239, K249-A250, and R258-M2598). gel electrophoresis. Proteomics 2003;3:832–846. Further, Liu, et al.9) immunochemically confirmed the pro- 2) Manabe T, Mizuma H, Watanabe K. A non-denaturing posed cleavage sites and decided other cleavage sites at protein map of human plasma proteins correlated with a denaturing polypeptide map combining techniques of R443-T444, R512-H513, K527-T528, and K602-M603. The micro two-dimensional gel electrophoresis. Electrophore- results shown in Table 2 well coincide with these reports, sis 1999;20:830–835. suggesting that the minor spots detected on the Type-II 2- 3) Manabe T, Okuyama T. Analysis of cellular proteins by DE gels are the plasmin degradation products of the micro two-dimensional electrophoresis. In: Dunn MJ, edi- COOH-terminal two-thirds of the fibrinogen α-chains. tor. 2-D PAGE ’91. London: Zebra Printing, 1991:7–11. Although the physiological roles of the COOH-terminal 4) Manabe T, Okuyama T. Automatic gradient gel former for two-thirds of the fibrinogen α-chain have not been estab- micro slab gels and immobiline capillary gels. In: Dunn lished, Rudchenko, et al. 10) suggested that they are natu- MJ, editor. Electrophoresis ’86. Weinheim: VCH, 1986: rally occurring products of fibrinolytic system activation, 613–616. since they circulate together with intact fibrinogen and also 5) Oakley BR, Kirsch DR, Morris NR. A simplified ultrasen- they can serve as a cross-linking partner. The finding in our sitive silver stain for detecting proteins in polyacrylamide previous paper, that most of the fibrinogen α-chain frag- gels. Anal Biochem 1980;105:361–363. 1) 6) Manabe T, Jin Y. Assignment of human plasma polypep- ments apparently bound with IgG , might support this pro- tides on a nondenaturing 2-D gel using MALDI-MS and posal. However, the artificial degradation of fibrinogen α- PMF and comparisons with the results of intact protein chains shown in Figs. 1 and 2 must be prevented when one mapping. Electrophoresis 2007;28:843–863. aims the analysis of human plasma proteins in the absence 7) Pizzo SV, Schwartz ML, Hill RL, McKee PA. The effect of of denaturing agents. The micro gel system we employed in plasmin on the subunit structure of human fibrinogen. J this paper would facilitate the analysis of intact proteins, Biol Chem 1972;247:636–645. when the temperature is carefully kept around 4°C, 8) Takagi T, Doolittle RF. Amino acid sequence studies on because the time needed for the 2-DE separation of pro- the α chain of human fibrinogen. Location of four plasmin teins is only about 2 h. attack points and a covalent cross-linking site. Biochemis- try 1975;14:5149–5156. GRANTS 9) Liu CY, Sobel JH, Weitz JI, Kaplan KL, Nossel HL. Immu- This work was supported in part by a Grant-in-Aid for nologic identification of the cleavage products from the Aα- and Bβ-chains in the early stages of plasmin digestion Scientific Research (No.14540561) from the Ministry of of fibrinogen. Thromb Haematostasis 1986;56:100–106. Education, Science, and Culture, Japan. 10) Rudchenko S, Trakht I, Sobe JH. Comparative structural α REFERENCES and functional features of the human fibrinogen C domain and the isolated αC fragment. J Biol Chem 1996; 1) Manabe T, Yamaguchi N, Mukai J, Hamada O, Tani O. 271:2523–2530. Detection of protein-protein interactions and a group of