3. PURIFICATION AND BIOPHYSICAL CHARACTERIZATION OF SECRETED
3.4 PROTEIN SEQUENCING BY MALDI-TOF MS/MS
3.4.3 Results
The amino acid composition and identification of the purified enzyme was achieved by mass spectrometry (MALDI-TOF). Two main problems were encountered during sequencing: - low protein yield and lack of homogeneity in the enzyme sample. The purified enzyme gave a single band on SDS-PAGE gel (Figures 3.6B and 3.8A), but sequencing data revealed multiple proteins that were co-eluted with acid phosphatase (Table 3.8). Interesting peptides ranging from stress response proteins, hydrolases (carboxylases and proteinases) with most peptide sequences showing a strong homology to fungal enzymes. However, all data not related to the enzyme of interest, apase were not further analyzed. Peptides that showed a close match to acid phosphatase were selected and analyzed in details (Figure 3.15). The alignments, conducted by ClustalW and re-arranged by Vector NTI clearly demonstrated that apase from Cladonia portentosa is homologous to many fungal apases (Figure 3.16).
One of the peptides generated was MLAETNPA (peptide 9, Table 3.8). The fingerprint of this peptide fragmentation data at that region of the raw data was poor. The peptide was then assigned to the Swiss-Prot, MSDB and NCBInr protein sequence database. The hit came with histidine acid phosphatase with homology (69 %) to A. terreus accession number (Q0CVC9 (Figure 3.16A). The E score of 4 was obtained for this peptide and the match was not significant.
113 Table 3.8: Amino acid sequences from the purified acid phosphatase from Cladonia portentosa (not shown=#, shown*). Amino acid sequences from the purified acid phosphatase from Cladonia portentosa (not shown=#, shown*). Data analysis is based from SDS-gel excised from 3 bands corresponding to 55, 60 and 148 kDa.
Peptide no.
Amino acid Protein Estimated
size
Accession number
Match
1 EVAQLLSQSLATEAR Stress response protein Rds1 148 Q5B2D8 Aspergillus clavatus
2 DGLTGLQSTPR Putative uncharacterized protein 148 Q5B7K5 Aspergillus nidulans
3 TLPLGDSFP Unknown - - -
4 VLPQVLEATNR Carboxypeptidase 5 148 Q5VJG7 Sartorya fumigata
5 LLNNANLNR Unknown - - -
6 VYGPLLEMFR Unknown - - -
7 GAELLVPSPK Putative uncharacterized protein - - -
8 LAWQNFPTL Putative uncharacterized protein Q5B2D8 Emericella nidulans
9 MLAETNPA* Phytase 148 Apsergillus terreus
10 FLAETNPAPFGHP* Histidine acid phophatase/phytase 148 Q0CZR1 Aspergillus terreus
11 AVGLGYVEELLAR* Phytase 3 148 Aspergillus terreus
12 LVDGEEYGEVR Putative uncharacterized protein 148 Q0U704 Phaeosphaeria nodorum
13 SNELFTANLGSAR Aspartic proteinases 55
14 GSNPFDLLGDVFLK Aspartic proteinases /aspartic endopeptidase
55 Q9HF87 Botryotinia fuckeliana
15 AQGLGYVQEVLAR# Acid phosphatase 148 Aspergillus terreus
16 FGESGVWGFLNHLDSR Stress response gene Aspergillus nidulans
17 DALPVELD Unknown
18 SLYALFDQGNTR Aspartic proteinases 55kd
19 TFTQLAPMEAPR Unknown 60
20 LTSTTHTVNTDQR Galactose oxidases 60 Q4WH00 Aspergillus fumagatus
114 Figure 3.15: (A-C) MALDI-TOF peptide mass fingerprinting of acid phosphatase from Cladonia portentosa. A sample of the enzyme was resolved by SDS-PAGE and then subjected to in-gel digestion by modified trypsin. MALDI-TOF mass spectra were acquired in positive ionisation mode. The MASCOT database search retrieved peptide sequences: FLAETNPAPFGHG* and AVGLGYVEELLAR*
M/z
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
%
0 100 Zama gel band at 148kDa
080108 Zama 148 MaxEnt 3 200 [Ev-21158,It50,En1] (0.050,200.00,0.200,1400.00, 2,Cmp) 2: TOF MSMS 985.97ES+
(242.12) P H G F P A P N T E A L M (331.26) yMax
802.35
316.13
245.10
169.08
141.08
429.21
358.18 y1
634.27
500.23
629.29 796.30
y5
712.28 964.40
y7
916.40
844.34
1217.46
1146.48 1017.40
1078.43 y8
1379.50 y11 1308.53
y10
1645.70
1574.64 1380.59
1470.51 1927.61
1646.54 1831.64 1929.63 1999.67
M/z
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0 100 Zama gel band at 148kDa
080108 Zama 148 MaxEnt 3 331 [Ev-71326,It50,En1] (0.050,200.00,0.200,1400.00, 2,Cmp) 2: TOF MSMS 695.36ES+
R A L L E E V Y G L G V A yMax
1049.46 y9
228.11 b3
171.09 b2 143.10
a2 136.05
Y
829.41 730.34 y7
y6
313.19 a4 246.13
y2
601.32 y5 472.27 359.21 y4
y3 391.16 533.24
a6 691.25
632.30 a7
731.40
992.44 y8
830.33 943.64
1219.55 y11
1050.55 1201.52 1051.35
1371.63 1220.64
1221.44 1372.71
M/z
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
%
0 100 Zama gel band at 148kDa
080108 Zama 148 MaxEnt 3 200 [Ev-21158,It50,En1] (0.050,200.00,0.200,1400.00, 2,Cmp) 2: TOF MSMS 985.97ES+
(242.12) P H G F P A P N T E A L F (331.26) yMax
802.35
316.13
245.10
169.08
141.08
429.21
358.18 y1
634.27
500.23
629.29 796.30
y5
712.28 964.40
y7
916.40
844.34
1217.46
1146.48 1017.40
1078.43 y8
1379.50 y11 1308.53
y10
1645.70
1574.64 1380.59
1470.51 1927.61
1646.54 1831.64 1929.63 1999.67
A b so lu te in te n si ty ( %)
M/z
115 A) Peptide 9
Peptide 9 |Cladonia portentosa mLAETNPA 8 QOCVC9 |Aspergillus terreus mLAETNPA 565 QOCZR1 |Aspergillus terreus FLAqTNPA 540 A2R685 |Aspergillus niger FLAqTNPA 547
B) Peptide 10
Q0CVC9 |Aspergillus terreus mLAETNPAPFGq 565 Peptide10 |Cladonia portentosa FLAETNPAPFGh 12 Q0CZR1 |Aspergillus terreus FLAqTNPAPFGN 540 A2R685 |Aspergillus niger FLAqTNPAPFGN 547
C) Peptide 11
B2W4A5-1 |Pyrenophora tritici-repentis AiGIGYVeEvLAR 557 Q0U9E0 |Phaeosphaeria sodorum AiGVGYVqEvLAR 509 B2WJV4 |Pyrenophora tritici-repentis tQGVGYVNELLAR 473 Q0V0X4-1 |Phaeosphaeria sodorum tQGVGYVNELLAR 1001 Q5GGT6-1 |Neourospora crassa tQGVGYVNELLAR 596 Q7S9V5 |Neourospora crassa tQGVGYVNELLAR 493 B0DV43-1 |Laccaria bicolour vQGVGYVNELLAR 504 Q96VH9-1 |Peniophora lycii vQGVGYVNELLAR 473 Peptide11|Cladonia portentosa AvGlGYVeELLAR 13 Q0CZR1 |Aspergillus terreus AvGVGYVeELvAR 540 A6SFM5-1 |Botryotina fuckeliana AvGIGYVqEvvAR 469 B8PES5 |Postia placenta AQGIGYVqELvAR 546 A6RPE1 |Botryotina fuckeliana AQGlGYVqELLAR 507 A7EBV4 |Sclerotinia sclerotiorum AQGIGYVqELLAR 527
116 D)Peptide 15
Q7S9V5 |Neurospora crassa tQGVGYVNELLAR 596 Q5GGT6 |Neurospora crassa tQGVGYVNELLAR 497 B2WJV4 |Pyrenophora tritici tQGVGYVNELLAR 473 Q96VH9 |Peniophora lycii vQGVGYVNELLAR 439 B0DV43 |Laccaria bicolor vQGVGYVNELLAR 504 B6GVW5 |Penicillium chrysogenum AQGIGYVeEvaAR 508 B2WJV4 |Pyrenophora tritici AiGIGYVeEvLAR 557 A6SFM5 |Botryotinia fuckeliana AvGIGYVqEvvAR 468 Q0U9E0 |Phaeosphaeria nodorum AiGVGYVqEvLAR 509 B8PES5 |Postia placenta AQGIGYVqELvAR 546 A7EBV4 |Whetzelinia sclerotiorum AQGIGYVqELLAR 527 Peptide15|Cladonia portentosa AQGlGYVqEvLAR 13 A6RPE1 |Botryotinia fuckeliana AQGlGYVqELLAR 506
Figure 3.16: (A-D) Multiple alignment of peptides obtained from sequencing of acid phosphatase from C. portentosa. The peptides (underlined) were searched using the expasy search engine to include proteins found in the UniProtKB/TrEMBL. Pam30 was used as comparing matrix in combination with gap alignment. The matching sequences were aligned using alignX and later rearranged using Vector NTI (Invitrogen). The position of the amino acid within each database entry is shown. The alignment of amino acid sequences of acid phosphatase from various sources is indicated. The accession number (Acc no.) and name of the species from which the acid phosphatase gene was cloned or purified is shown.
The length indicates the total size of the protein (in amino acids- as stated in the database entry). The different colours indicate area of homology.
Identical amino acids are shaded in blue, similar amino acids are in gray and no colour=no homology.
117 To get a „convincing result,‟ the sequencing was repeated and a longer peptide (10) (Figure 3.16B) FLAETNPAPFGHP was obtained (Figure 3.16). Interestingly, this peptide was similar to peptide 9. However, in the first peptide, the initial M/F alternate calls were considered valid only if methionine was oxidised. Methionine oxide and phe (F) are the same mass but a 'normal' methionine would not fit the data, however, the oxidation of methionine is quite common. What made this more difficult was that both peptides can act homologous to Aspergillus terreus and both these sequences FLAETNPAPFGHP and MLAETNPAP belong to histidine acid phosphatase, which codes for two different proteins in the same organism (accession number QOCZR1 and QOCV9, respectively) (Table 3.9). The E score of 0.76 suggests that peptide 10 was homologous to histidine acid phosphatase from A. terreus (75%), followed by phytase from A. niger (74%) (Table 3.9).
Table 3.9: Identified FLAETNPAPFGHG peptide 10 from MALDI-TOF MS/MS analysis were searched against the NCBI database using expasy website. The peptide was queried by blastp in the UniProt Knowledgebase (Swiss-Prot + TrEMBL). Comparison matrix was conducted by PAM30 using a gap alignment.
Accession number
Organism Gene name Score E-value %
Q0CZR1 Aspergillus terreus Histidine acid phosphatase 0.76 75
A2R685 Aspergillus niger Phytase A 0.76 73
Q0CVC9 Aspergillus terreus Acid phosphatase 1.0 74
A strong signal yielding a peptide sequence of AVGLGYVEELLAR (peptide 11) was generated (Figure 3.16C). The peptide identity was searched using expasy database, and blasted with known proteins in the NCBI data base. Comparison with the sequence data base using a gap alignment analysis indicated that the hypothetical protein had the highest homology (75 % identity) to a histidine acid phosphatase protein (GenBank accession number Q0CZR1) from A.
terreus (Table 3.10). This protein also shares 73 % homology to Botryotinia fuckeliana apase protein (GenBank accession number A6RPE1) and shares 71 % identity to Pyrenophora tritici- repentis phytase gene (GenBank accession number B2WEA5). The E value score indicates that this peptide is closest to A. terreus (Table 3.10). A significant E value score of 0.06 was obtained.
118 Table 3.10: Identified AVGLGYVEELLAR peptide from MALDI-TOF MS/MS analysis was searched against the NCBI database using the expasy website: The peptide was queried by blastp in the UniProt Knowledgebase (Swiss-Prot + TrEMBL). Comparison matrix was conducted by PAM30 using a gap alignment. Histidine acid phosphatase =HAP.
Accession number
Organism Gene name Score E-
value
%
Q0CZR1 Aspergillus terreus HAP 0.06 75
A6RPE1 Botryotinia fuckeliana Acid phosphatase 0.11 73
B2W4A5 Pyrenophora tritici Phytase A 0.19 71
A7EBV4 Sclerotinia sclerotorum HAP 1.1 65
Q0U9E0 Phaeosphaeria nodorum Acid phosphatase 2.0 63
Q0U9E0 Postia placenta Phytase A 3.7 61
Q96VH9 Peniophora lycii Phytase A 3.7 61
Q7S9V5 Neurospora crassa Phytase A 3.7 61
Q5GGT6 Neurospora crassa Phytase B 3.7 61
B2WJV4 Pyrenophora tritici- repentis 3-phytase B 3.7 61
B0DV43 Laccaria bicolor Acid phosphatase 43.7 60
There was a significant homology between the peptide 15 „AQGLGYVQEVLAR‟ of acid phosphatase and histidine acid phosphatase protein from Botryotinia fuckeliana (GenBank accession number A6RPE1 and Sclerotinia sclerotiorum (A7EBV4) (Figure 3.16D). The E value score was 0.003, indicating the most significant hit (Table 3.11). This protein also shares 77 % identity to Sclerotinia sclerotiorum apase protein (GenBank accession number A7EBV4) and shares 71 % identity to Phaeosphaeria nodorum gene (GenBank accession number Q0U9E0). Other proteins were homologous to these peptides but the E scores were above 1, thus, were considered not significant (Table 3.11).
Interestingly, this peptide under expasy matched Botryotinia fuckelinia. In BLAST output, this peptide was translated as a hypothetical protein called a phytase, with the closest match Sclerotinia. This peptide suggests that the whole protein is also probably an acid phosphatase, but does not seem to be in related with peptide 10 and 11 but probably related to the second apase which was present in low quantity in some samples, which was only detected under ion- exchange chromatography.
119 Table 3.11: The AQGLGYVQEVLAR peptide was searched using the expasy search engine to include proteins found in the UniProtKB/TrEMBL. Comparison matrix was conducted by PAM30 using a gap alignment.
Accession number
Organism Gene name Score E-
value
%
A6RPE1 Botryotinia fuckeliana HAP 0.003 85
A7EBV4 Sclerotinia sclerotiorum Acid phosphatase 0.033 77
Q0U9E0 Phaeosphaeria nodorum Acid phosphatase 0.19 71
B8PES5 Postia placenta Phytase A 0.47 68
B6GVW5 Penicillium chrysogenum Phytase 0.85 66
B2W4A5 Pyrenophora tritici Phytase A 1.1 65
A6SFM5 Botryotinia fuckeliana Histidine acid phosphatase 2.0 63
Q96VH9 Peniophora lycii Phytase A 6.6 59
Q7S9V5 Neurospora crassa Phytase A 6.6 59
Q5GGT6 Neurospora crassa Phytase B 6.6 59
B2WJV4 Pyrenophora tritici-repentis 3-Phytase B 6.6 59
B0DV43 Laccaria bicolor Acid phosphatase 6.6 59
120