Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3647-3652, April 1996 Biochemistry
Mass spectrometric amino acid sequencing of a mixture of seed storage proteins (napin) from Brassica napus, products of a multigene family PETER M. GEHRIG*t, ANDRZEJ KRZYZANIAKI, JAN BARCISZEWSKIt, AND KLAUS BIEMANN*§ *Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; and :Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland Contributed by Klaus Biemann, December 22, 1995
ABSTRACT The amino acid sequences of a number of four other points, yielding two disulfide-linked peptide chains closely related proteins ("napin") isolated from Brassica with molecular masses in the range of 4-10 kDa (2). napus were determined by mass spectrometry without prior Several different napin polypeptides can be expected as separation into individual components. Some ofthese proteins products of expression of a gene family of at least 10 (4) and correspond to those previously deduced (napA, BngNAP1, and perhaps more than 16 members (5). Four genomic sequences, gNa), chiefly from DNA sequences. Others were found to differ napA (4), napB (6), gNa (5), and BngNAP1 (12), and three to a varying extent (BngNAP1', BngNAPIA, BngNAPlB, cDNA sequences, pNl and pN2 (3), and pNAP1 (2), were BngNAP1C, gNa', and gNaA). The short chains of gNa and published. Mature napin protein was sequenced by Edman gNa' and of BngNAP1 and BngNAP1' differ by the replace- degradation without prior separation of isoforms (2). The ment of N-terminal proline by pyroglutamic acid; the long sequence of one single main component was found to corre- chains of gNaA and BngNAPlB contain a six amino acid spond to the cDNA sequence ofpNAP1 and the gene sequence stretch, MQGQQM, which is present in gNa (according to its of napA (which are identical), and only minor sequence DNA sequence) but absent from BngNAP1 and BngNAPlC. heterogeneity was observed. These alternations of sequences between napin isoforms are In another study, the isolation of five napin proteins from B. most likely due to homologous recombination of the genetic napus was described (13). The amino acid compositions of the material, but some of the changes may also be due to RNA three major components, napins nII, nIII, and nIV, the editing. The amino acids that follow the untruncated C termini sequences of the N- and C-terminal ends of both chains (14), of those napin chains for which the DNA sequences are known and the sequence of the entire small chain of nIII were (napA, BngNAPI, and gNa) are aromatic amino acids. This However, these could not be that the of the to the reported (15). napin proteins suggests processing proprotein leading unambiguously correlated with known gene or cDNA se- C termini of the two chains is due to the action of a protease and the results obtained for the sites of the that cleaves a bond. quences, processing specifically G/S-F/Y/W napin chains were only partially consistent with those proposed previously (2). Napin, a member of the 2 S albumin class of proteins, is one To assess the function of plant storage proteins and even- of the major seed storage proteins in Brassica napus, consti- tually allow genetic engineering of improved grain crops, the tuting about 20% of the total protein content in mature rape detailed primary structure of these proteins must be known. seeds (1, 2). These proteins are expressed during seed devel- For this reason, we have carried out, mainly by mass spec- opment as precursors, undergo co- and posttranscriptional trometry, the complete characterization of the amino acid modifications, and are then transported to membranous or- and the truncated termini of all abundant where in sequences relatively ganelles (protein bodies) they accumulate large 14-kDa napin forms from B. napus. Because of the high degree quantities. There is increasing interest in napin proteins and of variants and due to their their because a model for homology among napin ragged genes they represent good studying ends, and isolation of all isoforms are difficult. For both the expression of a multigene family and protein matu- separation ration in cells studies this reason, and in order to obtain a complete picture of the processes plant (3-7). DNA-binding the structural was done on some evidence for a role of in the of expressed napin proteins, analysis provide napin regulation in one its own level of and the mixture of the 14-kDa napins that all eluted HPLC high synthesis (A.K. J.B., unpublished a different mass data). Napin contains a short track rich in basic amino acids peak. Using strategy involving spectrometric typical of nuclear localization signals (8), and also glutamine- methods, napin protein sequences corresponding exactly to rich domains that are characteristic of one group of transcrip- known DNA sequences, as well as others differing by a few tion factors (9, 10). Furthermore, x-ray studies of phaseolin, amino acids, were identified and their expression and process- another seed storage protein, showed a domain with structural ing patterns were analyzed in detail. The environment of the similarity to the helix-turn-helix motif found in certain DNA- N and C termini of different isoforms showed common structural binding proteins (11). features indicating processing by one or two specific proteases. Mature napin consists of two polypeptide chains that are linked by disulfide bonds (2). It appears from comparison of MATERIALS AND METHODS these polypeptide chains with the corresponding DNA se- quences that the initial translation product is a 20-kDa pre- Isolation of Napin. Rape seeds (B. napus var. Bor; 500 g) cursor protein (2-4) that contains both the peptide chains of were homogenized and extracted with buffer A (50 mM mature napin as well as peptide stretches that are removed during maturation. First, an N-terminal signal peptide is Abbreviations: MALDI, matrix-assisted laser desorption ionization; removed from the precursor, which is subsequently cleaved at TOF, time-of-flight; MS, mass spectrometer(try); PSD, postsource decay; RP-HPLC, reversed-phase HPLC; TFA, trifluoroacetic acid; CID, collision-induced dissociation. The publication costs of this article were defrayed in part by page charge tPresent address: Brain Research Institute, University of Zurich, payment. This article must therefore be hereby marked "advertisement" in August Forel-Strasse 1, CH-8029, Zurich, Switzerland. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 3647 Downloaded by guest on September 27, 2021 3648 BiceityGergeal Proc. Natl. Acad. Sci. USA 93 (1996) NaH2PO4, pH 7.0/1 mM EDTA) and centrifuged for 1 h [all the napin chains, sinapinic acid in acetonitrile/water/TFA centrifugations were at 4000 rpm (2504 x g)]. To 400 ml of (30:70:0.1) was used as the matrix and the TOF MS was supernatant, solid (NH4)2SO4 was added to 30% of saturation operated in the reflector mode at an accelerating potential of and centrifuged. The supernatant was again brought to 75% 27.5 kV. The instrument was internally calibrated with melittin saturation of (NH4)2SO4 and centrifuged; the pellet was then and bovine insulin, or insulin and cytochrome c as standards. dissolved in 50 ml of buffer A, dialyzed against the same buffer, Mass spectra were generated from 50-100 single laser shots. and passed over a Sephadex G-50 column. Fractions containing The MALDI-TOF mass spectra of enzymatic digests were napin were collected, precipitated with (NH4)2SO4 (75% of measured with a linear VT 2000 MALDI-TOF MS (Vestec, saturation), and centrifuged. Crude napin was dissolved in buffer Houston) equipped with an N2 laser and operated in the B (same as buffer A, but pH 7.4), dialyzed against the same buffer, positive-ion mode at an accelerating voltage of 30 kV. The loaded onto a Sephadex 50 coarse medium column, and eluted matrix solution was a-cyano-4-hydroxycinnamic acid in aceto- with a gradient of 0.15-0.35 M NaCl in buffer B. Fractions nitrile/H20 (50:50). After initial external calibration with containing napin were pooled, precipitated with (NH4)2SO4, known standards on the same target, the spectra were cali- redissolved in buffer A, and dialyzed. Finally, 1.4 g of crude napin brated internally using previously identified peaks. The mass was obtained. For the work described here, 5 mg was further spectra were the sum of 20-30 laser shots. purified by reversed-phase (RP)-HPLC on an Aquapore-C4 MALDI-postsource decay (PSD) mass spectra (18) were column (Brownlee Lab) using a gradient of 0-80% acetonitrile acquired with a Bruker (Billerica, MA) Reflex model instru- in H20 containing 0.05% trifluoroacetic acid (TFA). ment equipped with an N2 laser for MALDI. The accelerating Reduction and S-Ethylpyridylation of Napin. Napin (0.28 voltage was 28.5 kV and the final reflector lens element was at mg, 20 nmol) was dissolved in 100 utl NH4HCO3 buffer (0.01 30 kV. For the recording of PSD spectra, the reflector M, pH 8.3), reduced, and S-ethylpyridylated by a 10-fold excess potential was lowered to 0.95 kV in 13 steps. Further details are of triethylphosphine (Aldrich; 1 wt-% in 2-propanol) and a described elsewhere (19). 100-fold excess of 4-vinylpyridine (Sigma). The mixture was Fast Atom Bombardment and Tandem Mass Spectrometry. incubated under argon at 37°C for 2 h and then lyophilized. Mass spectrometry was carried out on a JEOL HX1 10/HX110 Enzymatic Digestion of Napin. S-Ethylpyridylated napin instrument as described (20, 21), except that a Cs+ ion gun was (0.22 mg, 16 nmol) was sequentially digested with Endo-Lys-C used for ionization and the collision energy was 7 keV. (Wako Pure Chemical, Osaka), 150:1 substrate:enzyme, at Edman Sequencing. Automated Edman sequencing was 30°C for 6 h followed by trypsin (Boehringer Mannheim), 100:2 carried out at the Massachusetts Institute of Technology at for 3 h in 100 buffer substrate:enzyme, 37°C ,tl NH4HCO3 Biopolymer Laboratory using an Applied Biosystems model (0.01 M, pH 8.3). S-Alkylated napin (0.11 mg, 8 nmol) was 477A sequencer and model 120 on-line PTH amino acid analyzer. digested with 2 wt-% of a-chymotrypsin in 250 ,tl NH4HCO3 buffer (0.1 M, pH 7.8) using otherwise identical conditions as for trypsin,'and another 0.22 mg (16 nmol) was treated with 2 RESULTS buffer and wt-% of Endo-Glu-C in 0.1 M sodium phosphate To facilitate the discussion, it may help to summarize the final 0.02% sodium azide (Pierce) at 37°C for 2 h. Finally, 0.22 mg conclusion first. It turned out that the napin isolated from B. (16 nmol) S-alkylated napin was digested with 2 wt-% of is a rather mixture of similar at napus complex, inseparable very thermolysin in 100 ,/l of NH4HCO3 buffer (0.01 M, pH 8.3) The MALDI mass of this mixture exhibits were fractionated peptides. spectrum 37°C for 2 h. These digests partially by a resolved from 13 to 15 mass broad, only partially signal ranging RP-HPLC for spectrometric analyses. kDa. To make the it was to small amounts of HPLC- problem manageable, necessary Relatively (<1 nmol) partially convert the native material into the short and long chains by fractionated (see Fig. 1), S-ethylpyridylated napin chains were reduction and alkylation of the disulfide bonds (2). The digested with 1 wt-% of Endo-Lys-C in 100 ,/l NH4HCO3 the MALDI of which shows buffer M, at 30°C for 6 h and also with 6 wt-% resulting mixture, spectrum (0.01 pH 8.3) clusters of peaks around 4 kDa and 10 kDa, can be partially of clostripain (Sigma) in 60 ,l of NH4HCO3 buffer (0.01 M, pH RP-HPLC The in the MALDI 1 mM of at 37°C for separated by (Fig. 1). signals 7.8) containing 1,4-dithiothreitol (Pierce) of fractions 1-6 are summarized in Table 1. These data were matrix-assisted spectra 3 h. These digests directly analyzed by to that the laser ionization-mass and experiments described below led the conclusion desorption spectrometry (MALDI-MS) more of the native material are com- fractionation. All were carried out abundant components without prior digestions posed of the six short chains and five long chains depicted in under argon. chain HPLC. Partial fractionation of reduced and S-ethylpyridy- Fig. 2 (there was no evidence for the presence of the long lated chains and of obtained from of gNa, the sequence of which is shown here for comparison napin peptides enzymatic The mixture is further N-terminal cleavage was carried out by RP-HPLC (214 nm detection) on only). complicated by truncations of the short chains and N- or C-terminal truncation a Vydac C18 column (25 cm x 4.6 mm i.d.), at a flow rate of 1 ml/min (see Fig. 1). Solvent A is 0.05% TFA in water; solvent of the long chains (Table 1). Most of the information that led B is 0.035% TFA in acetonitrile. chains were fraction- to these assignments is based on the digestion of each of these Napin with and The ated in a linear gradient from 95% A/5% B to 55% A/45% B six HPLC fractions Endo-Lys-C clostripain. [M + are in Tables 2 and 3 for the over 50 min. The gradient used for digests was typically 100% H]+ ions observed listed A to 60% A/40% B over 40 min. For fast-atom bombardment Endo-Lys-C and in Tables 4 and 5 for the clostripain digests. are on DNA-derived MS, 5-6 tul of 50% glycerol in water was added, followed by The assignments based the published lyophilization. MALDI-MS. Mass spectrometry was as in ref. 16. The were molecular weights of intact napins and of napin chains .2- measured on a Voyager MALDI-time-of-flight (TOF) MS (PerSeptive Biosystems, Framingham, MA) equipped with a 337-nm N2 laser (Laser Science, Newton, MA) and an ion 0-.r was 6 fraction# reflector. The spectrum of intact napin acquired in the I711111 TI 111Inrr1 T1 r IIIII nT IIr1im 11I1I ir l r1I1In II1nI positive-ion linear mode and with delayed ion extraction (17) min 28 30 32 34 36 38 40 42 44 46 at an accelerating potential of 20 kV. Ferulic acid in acetoni- trile/water (30:70) and 0.1% TFA was used as the matrix and FIG. 1. RP-HPLC trace of reduced and S-ethylpyridylated napin horse heart cytochrome c was used for external calibration. For indicating the regions where fractions 1-6 were collected. Downloaded by guest on September 27, 2021 Biochemistry: Gehrig et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3649
Table 1. Summary of MALDI mass spectral data of S-ethylpyridylated short and long napin chains HPLC Protein Calc. Measured fraction* chain Sequence Position [M + H]+ [M + H]+ 1 napA IP...QSGGGPS 7-39 3989.8 3991.4 napA RIP...QSGGGPS 6-39 4145.8 4147.6 2 napA GPFRIP...QSGGGPS 3-39 4447.2 4448.5 2 2 napA AGPFRIP...QSGGGPS 2-39 4518.3 4520.2 napA SAGPFRIP...QSGGGPS 1-39 4605.3 4604.3 2 BngNAP1/1' IP...QSGSGPS 7-39 3991.8 3991.4 1 BngNAPl/1' RIP...QSGSGPS 6-39 4147.8 4147.6 1 BngNAP1/ GPFRIP...QSGSGPS 3-39 4449.1 4448.5 2 BngNAP1/1' AGPFRIP...QSGSGPS 2-39 4520.2 4520.2 1 BngNAP1 PAGPFRIP...QSGSGPS 1-39 4617.3 4617.4 2 BngNAPI' QAGPFRIP...QSGSGPS 1-39 4631.3 4631.4 BngNAP1A IP...QSGSGPS 7-39 4019.8 4019.1 BngNAP1A RIP...QSGSGPS 6-39 4175.8 4175.8 2 BngNAP1A GPFRIP...QSGSGPS 3-39 4477.1 4477.6 BngNAP1A PAGPFRIP...QSGSGPS 1-39 4645.3 4645.3 1 gNa/gNa' RIP...GGGSGPS 6-41 4300.0 4299.3 2 gNa PAGPFRIP...GGGSGPS 1-41 4769.5 4769.1 2 gNa'
Short chains: Table 3. [M + H]+ ions of peptides obtained from Endo-Lys-C digestion of fractions 3-6 (long chains) of Fig. 1 1 10 20 30 40 napA (D)SAGPFRIPKCRKEFQQAQHLRACQQWLHKQAMQSGGGPS(W) BngNAP1 (N)P ...... K ...... S...(W) HPLC Measured Calc. Position* BngNAP1' 1902.4 1902.2 55-70 - Long chains: 3426.2 3425.1 6-31 - - 3949.4 3949.4 1-31 1 10 20 30 40 4 1773.2 1773.2 - 71-84 - napA (N)PQGPQQRPPLLQQCCNELHQEEPLCVCPTLKGASKAVKQQI-QQQ BngNAP1 (N) ...... VR... 3424.8 3425.1 - - 6-31 - BngNAP1B ...... 3550.8 3550.1 39-70 - BngNAPlC ...... VR ... 3949.7 3949.7 - 1-31 - gNaA gNa (N) 50 60 70 80 90 6 1773.2 1773.2 - 71-84 - 71-84 napA GQQQ-G--KQQMVSRIYQTATHLPKVCNIPQVSVCPFQKTMPGPS(Y) 3424.7 3425.1 6-31 - 6-31 BngNAP1 ...--. QQL..VI ...... (Y) 3519.8 3520.0 - 39-70 - 39-70 BngNAPlB .--MQ.QQM.HVI ...... - - BngNAPlC ...--.QQL..VI ...... 3949.7 3949.5 1-31 gNaA ...MQ.QQM..VI ...... R... .R...I...... G 3978.7 3979.7 - - 1-31 gNa ...MQ.QQM ...... R... I ...... G(F) Molecular weights of the protonated peptide ions were determined by MALDI-MS. FIG. 2. Aligned, full-length sequences of short and long napin *Positions are those corresponding to the sequences of heavy chains: chains. Only those amino acids that differ from napA are listed. Amino 6, napA; 7, BngNAP1; 8, BngNAP1B; 9, BngNAPlC; and 10, gNaA. acids in parentheses are those that precede or follow the processed See Fig. 1. chains according to the published DNA sequences. Because the N-terminal amino acid of the short chains of 2020.3 derived from the C terminus of napA (Ser-36 vs. Gly-36, BngNAP1, BngNAP1A, and gNa is proline, the mass differ- A = 30 Da). ence of + 14 Da could possibly also be due to N-methylproline, The presence of the short chain of gNa is shown by an [M a modification that to our knowledge has been observed only + H]+ ion of m/z 4769.1 (Table 1) and the expected C- once (22). However, the MALDI-PSD spectrum mentioned terminal (22-41) clostripain peptide, [M + H]+ = m/z 2174.3 above matches only that of synthetic 1129.7 1129.3 - 13-21 - 1 722.1 721.9 7-11 7-11 7-11 7-11 7-11 1622.9 1621.9 - 10-21 - 1285.5 1285.5 12-21 t 12-21 12-21 12-21 - 2257.6 2257.6 13-29 - 13-29 13-29 13-29 2020.1 2020.3 22-39 2 511.5 511.7 10-12 10-12 10-12 10-12 10-12 2050.3 2050.3 22-39 - 814.7 815.0 3-9 3-9 3-9 3-9 3-9 2174.3 2174.5 22-41 22-41 886.1 886.0 2-9 2-9 2-9 2-9 2-9 3288.7 3288.7 - 12-39 972.8 971.1 1-9 2 644.8 644.8 1-6 1-6 1-6 983.0 983.2 1-9 1-9 1-9 658.4 658.8 - - - - -6t 996.9 997.2 1-9t 721.9 721.9 7-11 7-11 7-11 7-11 7-11 - 1119.3 1119.3 - 22-29 - -- 1285.4 1285.5 12-21 12-21 12-21 12-21 - - 1129.4 1129.3 - 13-21 - 2020.8 2020.3 22-39 - - 1621.8 1621.9 - 10-21 - 2050.7 2050.3 22-39 -- 2257.6 2257.6 13-29 - 13-29 13-29 13-29 2175.1 2174.5 22-41 22-41 3288.7 3288.7 - 12-39 Molecular weights of the protonated peptide ions were determined by MALDI-MS. Molecular weights of the protonated peptide ions were determined *Positions are those corresponding to the sequences of light chains: 1, by MALDI-MS. napA; 2, BngNAP1; 3, BngNAP1A; 4, gNa; and 5, gNa'. See Fig. 1. *See legend of Table 2 for light chain sequence positions. See Fig. 1. tm/z value does not fit any peptide of this chain. tm/z value does not fit any peptide of this chain. tAlso from BngNAPI'. tAlso from BngNAPI'. Downloaded by guest on September 27, 2021 Biochemistry: Gehrig et aL. Proc. Natl. Acad. Sci. USA 93 (1996) 3651 Table 5. [M + H]+ ions of peptides obtained from clostripain terminus. Edman sequencing of fraction 6 indeed revealed the digestion of fractions 3-6 (long chains) of Fig. 1 presence of both glycine and serine at position 3, which causes the observed mass difference of -30 Da. This finding explains HPLC Measured Calc. Position* a similar earlier observation (14). fraction [M + H]+ [M + H]+ 6 7 8 9 10 The peptides produced from fraction 4 by clostripain (Table 3 810.7 810.9 1-7 -- 5) are almost identical to those from BngNAP1, with the 2130.1 2129.4 39-60 - - - exception of m/z 1885.7 (fraction 4) vs. m/z 1856.1 (fraction 2428.3 2427.8 36-60 - - 6). Because the latter corresponds to peptide 43-58 of Bng- 3311.4 3313.0 61-88 - - NAP1, the former most likely represents the same region but 3409.6 3410.1 61-89 - - differs in mass by +29.6 Da. A similar difference (+31.0 Da) 3499.2 3497.1 61-90 - - is observed between the corresponding Endo-Lys-C peptides 3501.2 8-35 - - of m/z 3519.8 (from BngNAP1, fraction 6) and m/z 3550.8 in 4 810.6 810.9 - 1-7 fraction 4 (Table 3). Finally, the set of [M + HI+ ions also 829.0 829.0 - 36-42 - differs by an average of 30.1 Da. All these data point to the 1885.7 1885.1 43-60 - region 43-60 for the sequence difference. While the peptide 3313.9 3313.0 - 61-88 - ion of m/z 1885.7 is a minor component in the clostripain 3410.8 3410.1 61-89 - digest, more of it had also been isolated from an Endo-Lys- 3500.9 3497.2 - 61-90 - C/trypsin digest. A combination of micro-Edman sequencing (in 3501.2 - - 8-35 the presence of a considerable excess ofpeptide IYQTATHLPK, 4293.9 4293.1 - 1-35 - - m/z 1171.8) and MALDI-PSD data suggested the sequence 4311.0 4311.2 8-42 QQQGMQGQQMQHVISR for the segment 43-60 (Table 6) of 5 709.2 709.9 - --- 71-75 this new napin, which we designate BngNAP1B. 828.8 829.0 - - 36-42 Fraction 5 also seems to represent a new long chain and its 953.2 953.0 - --- 0-7 N-truncated analogues. The N-terminal clostripain peptide 1200.6 1200.4 - - - - 61-70 0-7 of [M + H]+ = m/z 953.2 (Table 5, fraction 5) as well as 1695.7 1696.1 - 76-90 peptides 43-60, 71-75, and 76-90 fit the amino acid sequences 2132.4 2132.4 - - - - 43-60 of the corresponding portions of gene gNa (5). However, 3500.6 3501.2 - - - - 8-35 clostripain peptides [M + H]+ = m/z 828.8 and m/z 4307.7 4307.7 4311.2 - - - 8-42 seem to match m/z 828.2 and m/z 4310.4 in the same digest of 4432.9 4435.2 - - - - 0-35 BngNAP1 (fraction 6), corresponding to peptides 36-42 and 6 810.7 810.9 - 1-7 8-42, respectively. From the latter, we conclude that in this 828.2 829.0 - 36-42 - 36-42 napin, which we term gNaA, amino acids 10 and 11 are both 1856:1 1855.0 - 43-60 - 43-60 leucine (as in BngNAP1) and not proline and that the amino 3314.4 3313.0 - 61-88 - 61-88 acid at position 38 is lysine and not arginine, as the gene 3410.0 3410.1 - 61-89 - 61-89 sequence of gNa would require. This is further corroborated by 3500.7 3497.2 - 61-90 - 61-90 the Endo-Lys-C peptide, [M + H]+ = m/z 4091.1, obtained 3501.2 - 8-35 - 8-35 from fraction 5 (Table 3), which corresponds to the region 4310.4 4311.2 8-42 - 8-42 0-31 with Leu [10]-Leu [11], and by the presence of the Molecular weights of the protonated peptide ions were determined Endo-Lys-C peptide 39-84. This Leu-Leu placement is re- by MALDI-MS. dundantly proven by the detection of an Endo-Glu-C peptide *See legend of Table 3 for heavy chain sequence positions. See Fig. 1. 0-17, [M + H]+ of m/z 2289.4, calculated 2289.6, and a thermolysin peptide 0-10, [M + H]+ of m/z 1259.0, calculated The MALDI-MS data obtained from fraction 6 similarly 1260.5 (data not shown). The [M + H]+ ion of m/z 1200.6 of indicate that it consists chiefly of BngNAP1 (12), both full- the clostripain digest of fraction 5 does not match any known length C-terminally truncated (loss of S and PS) and N- napin DNA sequence. The high-energy CID spectrum of the terminally truncated (loss of PQGPQ) versions. There is also corresponding Endo-Lys-C/trypsin peptide indicates that it another isoform, termed BngNAP1C, that is 29.7 Da heavier represents aa 61-70 and that 61 is not valine, as the gene than BngNAP1. Pairs of peaks differing by about the same sequence of gNa would require, but isoleucine as in all other mass were also found for the C-terminally processed forms, but napin variants. The sequences (determined by CID-MS or not for those starting with amino acid 6. Thus, the only MALDI-PSD) of these and other proteolytic peptides that difference between these two isoforms must be near the N further confirm the assignment of the structures of the napin Table 6. Sequences (determined by CID or PSD) supporting new napin structures Measured [M + H]+ A* Sequence Napin Positiont Digest 996.5 0.0