Literature DB >> 29896512

Peptide data on the disulfide bond analysis of baculovirus produced Pfs25 by LC-MSMS.

Shwu-Maan Lee1, Jordan L Plieskatt1, C Richter King1.   

Abstract

This article contains the peptide data obtained while performing disulfide bond mapping of the recombinant Plasmodium falciparum protein, Pfs25, produced from the baculovirus expression system. Pfs25 is a malaria transmission-blocking vaccine candidate, with a compact and complex structure including 22 cysteines. This supplementary data is related to the research "Disulfide bond mapping of Pfs25, a recombinant malaria transmission blocking vaccine candidate" (Lee et al., 2018) [1]. In brief, Pfs25 was digested with trypsin/Lys-C and derived peptides separated by High Performance Liquid Chromatography (HPLC) and analyzed by mass spectrometry (MS) by MSE fragmentation. The theoretical peptides and their respective masses along with disulfide bond locations with linked peptides are presented here alongside the mass spectrometry analysis. The raw mass spectrometry data is made available through the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982.

Entities:  

Keywords:  Disulfide; LC-MSMS; Malaria; Mass spectrometry; Pfs25

Year:  2018        PMID： 29896512      PMCID： PMC5996233          DOI： 10.1016/j.dib.2018.03.034

Source DB:  PubMed          Journal:  Data Brief        ISSN： 2352-3409


Specifications Table Value of the data The derived peptides and mass spectrometry data is provided here for further details from the disulfide bond analysis of Pfs25. The disulfide bond locations and linked peptides are discussed alongside the mass spectrometry analysis here and data made accessible to the scientific community. Pfs25 is a compact and complex 17.9 kDa protein with 22 cysteines (11 disulfide bonds) that has presented difficulty in prior disulfide bond analysis A method was developed to map the disulfide bonds of a complex and compact protein, which may be applicable to other proteins, an important step in recombinant protein development for vaccines.

Data

The Pfs25 disulfide bond mapping peptides are discussed in further detail in this manuscript to further support the elucidation of disulfide bonds of Pfs25 as discussed in [1]. Further, the mass spectrometry RAW files have been deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE). Theoretical peptides, produced from Trypsin/Lys-C digestion of Pfs25, are presented in Table 1.
Table 1

Theoretical Fragments for Trypsin/Lys-C Digestion of Pfs25.

PeptidesPositionPeptide LabelTheoretical Mass (Da)
DAK1–3T1332.17
VTVDTVCK4–11T2863.44
R12-12T3174.11
GFLIQMSGHLECK13–25T41461.71
CENDLVLVNEETCEEK26–41T51865.80
VLK42–44T6358.26
CDEK45–48T7493.18
TVNKPCGDFSK49–59T81194.57
CIK60–62T9362.20
IDGNPVSYACK63–73T101165.54
CNLGYDMVNNVCIPNECK74–91T112027.86
QVTCGNGK92–99T12805.38
CILDTSNPVK100–109T131088.55
TGVCSCNIGK110–119T14980.44
VPNVQDQNK120–128T151040.53
CSK129–131T16336.15
DGETK132–136T17548.24
CSLK137–140T18449.23
CLK141–143T19362.20
ENETCK144–149T20722.29
AVDGIYK150–156T21764.41
CDCK157–160T22467.15
DGFIIDQESSICTHHHHHH161–179T232248.98
Theoretical Fragments for Trypsin/Lys-C Digestion of Pfs25. Utilizing Biopharmalynx 1.3 the mass spectral data was analyzed and compared to the theoretical peptides to obtain the localization of the 11 disulfide bonds present in the recombinant Pfs25. The disulfide bond locations and linked peptides (including theoretical and observed) masses are presented in Table 2. Each disulfide bond (referenced by nomenclature SS#) is further presented with the peptide information and mass spectrometry (MS) and MSMS data obtained during the analysis in the subsequent figures and tables presented in this manuscript.
Table 2

Disulfide bond locations for Pfs25 including theoretical and observed masses of fragments.

Disulfide BondLinked CysteinesTryptic Peptide LabelTheoretical Mass (Da)Observed Mass (Da)Mass Error (ppm)
SS1Cys10-Cys24T2+T42323.13752323.11529.6
SS2Cys26-Cys38T51863.78661863.77526.1
SS3Cys45-Cys60T7+T9853.3674853.36562.1
SS4Cys54-Cys72T8+T102358.06702358.07995.5
SS5Cys74-Cys85T11+T133112.37963112.35348.4
SS6Cys90-Cys100
SS7Cys95-Cys113T12+T14+T162117.93262117.91826.8
SS8Cys115-Cys129
SS9Cys137-Cys148T18+T201169.50561169.49707.3
SS10Cys141-Cys157T19+T22+T233074.30033074.27956.8
SS11Cys159-Cys172
Disulfide bond locations for Pfs25 including theoretical and observed masses of fragments.

Disulfide bond SS1

A total of 30 fragments were observed, with 20 fragment ions of this peptide consistent with the linkage of Cys10 and Cys24. The remaining ten fragment ions were consistent with constituent peptides (Table 3, Fig. 1).
Table 3

Disulfide bond SS1 (Cys10-Cys24) peptides.

AssignmentTheoretical Mass (Da)Observed Mass (Da)Mass Error (Da)Intensity (counts)Identification
30 FragmentsConstituent Peptides 10 Fragments1/a2173.129173.05680.072238VT
1/b5516.267516.26060.006369VTVDT
1/y1147.1133147.04150.071819K
2/a2177.1028177.10260.0002567GF
2/b2205.0977205.0983−0.0006691GF
2/b3318.1818318.1831−0.0013634GFL
2/b4431.2658431.18570.080182GFLI
2/b6690.3649690.36120.003740GFLIQM
2/b7777.3969777.4837−0.086834GFLIQMS
2/b9971.4773971.44250.034852GFLIQMSGH













Cys10 and Cys24 20 Fragments1/b7-2/y31094.4861094.4770.0095103VTVDTVC=ECK
1/b7-2/y91747.7821747.827−0.045486VTVDTVC=QMSGHLECK
1/y2-2/a121535.7171535.6750.0422196CK=GFLIQMSGHLEC
1/y2-2/y4739.3483739.3598−0.0115163CK=LECK
1/y2-2/y5876.4072876.39710.010168CK=HLECK
1/y2-2/z4722.3217722.22220.099526CK=LECK
1/y3-2/y3725.3326725.3351−0.0025101VCK=ECK
1/y3-2/y4838.4167838.32210.094656VCK=LECK
1/y4-2/y121852.9121852.8520.0603119TVCK=FLIQMSGHLECK
1/y5-2/y3941.4072941.39480.012550DTVCK=ECK
1/y5-2/y51191.551191.5050.045474DTVCK=HLECK
1/y5-2/y71335.6041335.5150.08847251DTVCK=SGHLECK
1/y5-2/y81466.6441466.662−0.0175177DTVCK=MSGHLECK
1/y7-2/y101907.9031907.8230.0797268TVDTVCK=IQMSGHLECK
1/y7-2/y21012.4811012.4380.0428181TVDTVCK=CK
1/y8-2/y102006.9712006.9470.0239390VTVDTVCK=IQMSGHLECK
1/y8-2/y112120.0552120.0460.0098112VTVDTVCK=LIQMSGHLECK
1/y8-2/y132324.1452324.1420.00294580VTVDTVCK=GFLIQMSGHLECK
1/y8-2/z101989.9451989.963−0.017863VTVDTVCK=IQMSGHLECK
1/z7-2/y2995.4542995.5009−0.046847TVDTVCK=CK
Fig. 1

Disulfide bond SS1.

Disulfide bond SS1. Disulfide bond SS1 (Cys10-Cys24) peptides.

Disulfide bond SS2

A total of 39 fragment ions of this peptide were observed with 36 fragment ions consistent with the linkage of Cys26 and Cys38. Three additional fragments were consistent with constituent peptides (Table 4, Fig. 2).
Table 4

Disulfide bond SS2 (Cys26-Cys38) peptides.

AssignmentTheoretical Mass (Da)Observed Mass (Da)Mass Error (Da)Intensity (counts)Identification
39 FragmentsConst. Pep.3 Fragments1/y1147.1133147.11320.00012129K
1/y2276.1559276.159−0.00311082EK
1/y3405.1985405.19720.0013825EEK













Cys26 and Cys38 36 Fragments1/b141589.6461589.6140.0321335CENDLVLVNEETCE (Internal)
1/y10-1/a31509.621509.5840.036687LVNEETCEEK=CEN
1/y10-1/b31537.6151537.644−0.0291246LVNEETCEEK=CEN
1/y10-1/b41652.6421652.644−0.0025544LVNEETCEEK=CEND
1/y10-1/b51765.7261765.7220.00434497LVNEETCEEK=CENDL
1/y11-1/b41751.711751.7090.00172109VLVNEETCEEK=CEND
1/y12-1/b11506.6821506.5870.094866LVLVNEETCEEK=C
1/y12-1/b21635.7251635.6310.0941586LVLVNEETCEEK=CE
1/y4-1/a121836.81836.7870.01272176CEEK=CENDLVLVNEET
1/y4-1/a2710.249710.2508−0.0018210CEEK=CE
1/y4-1/a61151.4711151.5−0.0284109CEEK=CENDLV
1/y4-1/a71264.5551264.470.0858499CEEK=CENDLVL
1/y4-1/b1609.2012609.2489−0.0477393CEEK=C
1/y4-1/b101634.7041634.6290.0752999CEEK=CENDLVLVNE
1/y4-1/b121864.7941864.797−0.002481665CEEK=CENDLVLVNEET
1/y4-1/b2738.2438738.2711−0.0273341CEEK=CE
1/y4-1/b3852.2868852.3066−0.0198172CEEK=CEN
1/y4-1/b4967.3137967.3153−0.0016992CEEK=CEND
1/y4-1/b51080.3981080.422−0.024317CEEK=CENDL
1/y4-1/b61179.4661179.4420.024380CEEK=CENDLV
1/y4-1/b71292.551292.4970.053258CEEK=CENDLVL
1/y4-1/b81391.6191391.5810.0372224CEEK=CENDLVLV
1/y5-1/a51153.4511153.4330.0179274TCEEK=CENDL
1/y5-1/b41068.3621068.366−0.0041172TCEEK=CEND
1/y5-1/b61280.5141280.5070.00651279TCEEK=CENDLV
1/y5-1/b81492.6661492.6070.05972TCEEK=CENDLVLV
1/y6-1/a61381.5621381.568−0.0067109ETCEEK=CENDLV
1/y6-1/b41197.4041197.41−0.00611266ETCEEK=CEND
1/y6-1/b51310.4881310.4880.0001885ETCEEK=CENDL
1/y6-1/b61409.5571409.5560.00091232ETCEEK=CENDLV
1/y7-1/a1940.3392940.3529−0.0137271EETCEEK=C
1/y7-1/b41326.4471326.4450.0021764EETCEEK=CEND
1/y7-1/b61538.5991538.5760.02261457EETCEEK=CENDLV
1/y8-1/a51525.5791525.593−0.0141104NEETCEEK=CENDL
1/y8-1/b41440.491440.497−0.00772676NEETCEEK=CEND
1/y8-1/b51553.5741553.575−0.00162348NEETCEEK=CENDL
Fig. 2

Disulfide bond SS2.

Disulfide bond SS2. Disulfide bond SS2 (Cys26-Cys38) peptides.

Disulfide bond SS3

A total of nine fragments were observed, with six fragment ions of this peptide consistent with the linkage of Cys45 and Cys60. Three fragment ions were consistent with constituent peptides (Table 5, Fig. 3).
Table 5

Disulfide bond SS3 (Cys45-Cys60) peptides.

AssignmentTheoretical Mass (Da)Observed Mass (Da)Mass Error (Da)Intensity (counts)Identification
9 FragmentsConst. Pep.3 Fragments1/y1147.1133147.115−0.0017163K
1/y2276.1559276.158−0.00284EK
1/y3391.1829391.2075−0.024647DEK
Cys45 and Cys60 6 Fragments1/a1–2/a2262.1048262.01830.086522C=CI
1/b1–2/a3421.0852421.1291−0.0439117C=CIK
1/b2-2/y3579.2271579.22610.00167CD=CIK
1/y4-2/a2680.2748680.2768−0.00223CDEK=CI
1/y4-2/b2708.2697708.2832−0.0135126CDEK=CI
1/y4-2/y3854.3752854.37480.00043305CDEK=CIK
Fig. 3

Disulfide bond SS3.

Disulfide bond SS3. Disulfide bond SS3 (Cys45-Cys60) peptides.

Disulfide bond SS4

A total of 68 fragments were observed, with 45 fragment ions of this peptide consistent with the linkage of T8 to T10 through Cys54 and Cys72. The remaining 23 fragments were consistent with constituent peptides (Table 6, Fig. 4).
Table 6

Disulfide bond SS4 (Cys54-Cys72) peptides.

AssignmentTheoretical Mass (Da)Observed Mass (Da)Mass Error (Da)Intensity (counts)Identification
68 FragmentsConstituent Peptides 23 Fragments1/a2173.129173.1297−0.00074507TV
1/a3287.1719287.09690.075897TVN
1/a4415.2669415.22240.044528TVNK
1/b2201.1239201.12350.00042754TV
1/b3315.1668315.08960.077251TVN
1/b4443.2618443.2626−0.0008248TVNK
1/y1147.1133147.06230.05116K
1/y2234.1454234.1455−0.0001700SK
1/y3381.2138381.20140.01241146FSK
1/y4496.2407496.24040.0003153DFSK
1/y5553.2622553.26040.0018564GDFSK
2/a3258.1454258.1545−0.009278IDG
2/a4372.1883372.1927−0.0044300IDGN
2/a5469.2411469.22780.013276IDGNP
2/a6568.3094568.30650.002991IDGNPV
2/a8818.4048818.36530.0396129IDGNPVSY
2/b1114.0919114.0410.050918I
2/b2229.1188229.119−0.00021962ID
2/b3286.1403286.1415−0.0012727IDG
2/b4400.1832400.1889−0.00571203IDGN
2/b5497.236497.2373−0.0013252IDGNP
2/b6596.3044596.3082−0.0038205IDGNPV
2/b7683.3364683.32060.0158211IDGNPVS













Cys54 and Cys72 45 Fragments1/a6-2/y51183.56041183.5410.01981608TVNKPC=SYACK
1/a6-2/y71379.68151379.6040.0773143TVNKPC=PVSYACK
1/a8-2/y71551.731551.6660.0636108TVNKPCGD=PVSYACK
1/a9-2/b101951.86821951.8230.045493TVNKPCGDF=IDGNPVSYAC
1/b10-2/a102038.90032038.99−0.0943TVNKPCGDFS=IDGNPVSYAC
1/b6-2/y3961.4599961.40650.0534124TVNKPC=ACK
1/b6-2/y81521.71941521.6740.0449161TVNKPC=NPVSYACK
1/b7-2/y71464.69791464.6750.022847TVNKPCG=PVSYACK
1/b8-2/y101865.81621865.827−0.010668TVNKPCGD=DGNPVSYACK
1/b8-2/y111978.90031978.8910.0092579TVNKPCGD= IDGNPVSYACK
1/b8-2/y21062.47121062.4410.0303364TVNKPCGD=CK
1/b8-2/y61482.67211482.6510.021137TVNKPCGD=VSYACK
1/b8-2/y71579.72491579.690.0347456TVNKPCGD=PVSYACK
1/b9-2/y51530.67211530.6670.0054180TVNKPCGDF=SYACK
1/b9-2/y71726.79321726.7410.052766TVNKPCGDF=PVSYACK
1/y11-2/y102246.02222246.0220.00052740TVNKPCGDFSK=DGNPVSYACK
1/y11-2/y112359.10622359.109−0.002757983TVNKPCGDFSK= IDGNPVSYACK
1/y11-2/y21442.67711442.6740.0034677TVNKPCGDFSK=CK
1/y11-2/y31513.71421513.7110.0028849TVNKPCGDFSK=ACK
1/y11-2/y41676.77761676.7770.0009999TVNKPCGDFSK=YACK
1/y11-2/y51763.80971763.8060.00371320TVNKPCGDFSK=SYACK
1/y11-2/y61862.87811862.893−0.0144201TVNKPCGDFSK=VSYACK
1/y11-2/y71959.93081959.9260.0056462TVNKPCGDFSK=PVSYACK
1/y11-2/y92130.99512131.013−0.0173418TVNKPCGDFSK=GNPVSYACK
1/y11-2/z102228.99562228.999−0.00371038TVNKPCGDFSK=DGNPVSYACK
1/y6-2/a101645.6991645.775−0.0762368CGDFSK=IDGNPVSYAC
1/y6-2/y111819.79941819.7950.004457CGDFSK= IDGNPVSYACK
1/y6-2/y41137.47081137.472−0.0007738CGDFSK=YACK
1/y6-2/y71420.6241420.6130.0114404CGDFSK=PVSYACK
1/y7-2/b101770.74671770.7220.024343PCGDFSK= IDGNPVSYAC
1/y7-2/y111916.85231916.8480.00393031PCGDFSK= IDGNPVSYACK
1/y7-2/y21000.42321000.424−0.0004627PCGDFSK=CK
1/y7-2/y31071.46031071.461−0.0007825PCGDFSK=ACK
1/y7-2/y51321.55571321.558−0.00241193PCGDFSK=SYACK
1/y7-2/y71517.67691517.681−0.00431667PCGDFSK=PVSYACK
1/y7-2/y91688.74121688.747−0.0057547PCGDFSK=GNPVSYACK
1/y8-2/y112044.94712044.9290.018264KPCGDFSK= IDGNPVSYACK
1/y8-2/y21128.51821128.5020.0162491KPCGDFSK=CK
1/y8-2/y41362.61871362.648−0.0288120KPCGDFSK=YACK
1/y8-2/y51449.65061449.6470.0038253KPCGDFSK=SYACK
1/y8-2/y81759.81471759.80.0151186KPCGDFSK=NPVSYACK
1/y9-2/y112158.99022158.9790.01121071NKPCGDFSK=IDGNPVSYACK
1/y9-2/y31313.59811313.580.0184279NKPCGDFSK=ACK
1/y9-2/y41476.66151476.6440.0173144NKPCGDFSK=YACK
1/z7-2/y2983.3967983.4453−0.0486113PCGDFSK=CK
Fig. 4

Disulfide bond SS4.

Disulfide bond SS4. Disulfide bond SS4 (Cys54-Cys72) peptides.

Disulfide bonds SS5 and SS6

A total of 90 fragments were observed and four fragment ions (1/b12, 1/b13, 1/b15, and 1/b16) were consistent with an internal disulfide bond linkage between Cys74 and Cys85. Thirty-three fragment ions were consistent with the linkage of Cys90 and Cys100. An additional 44 fragments of this peptide were consistent with the combined linkages of Cys74 to Cys85 and Cys90 to Cys100 and nine fragments consistent with constituent peptides (Table 7, Fig. 5).
Table 7

Disulfide bond SS5 (Cys74-Cys85) and SS6 (Cys90-Cys100) peptides.

AssignmentTheoretical Mass (Da)Observed Mass (Da)Mass Error (Da)Intensity (counts)Identification
90 FragmentsCys74 and Cys85 4 Fragments1/b121324.5131324.516−0.00311267CNLGYDMVNNVC (Internal)
1/b131437.5971437.5960.00063004CNLGYDMVNNVCI (Internal)
1/b151648.6921648.71−0.0175311CNLGYDMVNNVCIPN (Internal)
1/b161777.7351777.7280.007105CNLGYDMVNNVCIPNE (Internal)













Constituent Peptides 9 Fragments1/y1147.1133147.11230.0012902K
2/y2246.1818246.1842−0.0024789VK
2/y3343.2345343.15090.0836258PVK
2/y4457.2774457.27710.0003855NPVK
2/y5544.3095544.3134−0.0039711SNPVK
2/y6645.3572645.3581−0.00093696TSNPVK
2/y7760.3841760.37950.00462330DTSNPVK
2/y8873.4681873.46310.00512651LDTSNPVK
2/y9986.5522986.54940.0029381ILDTSNPVK













Cys90-Cys100 33 fragments1/a17-2/a72569.0652569.078−0.0127433CNLGYDMVNNVCIPNEC=CILDTSN
1/y18-2/b22240.9272240.983−0.0564114CNLGYDMVNNVCIPNECK=CI
1/y18-2/b42469.0382469.068−0.031446CNLGYDMVNNVCIPNECK=CILD
1/y18-2/b62657.1182657.1060.01290CNLGYDMVNNVCIPNECK=CILDTS
1/y2-2/a81063.4921063.4060.0859167CK=CILDTSNP
1/y2-2/b1351.1161351.204−0.087978CK=C
1/y2-2/b2464.2001464.2095−0.0094359CK=CI
1/y2-2/b3577.2842577.2840.0002142CK=CIL
1/y2-2/b4692.3112692.29970.0115322CK=CILD
1/y2-2/b7994.4338994.42780.006837CK=CILDTSN
1/y2-2/y101336.6611336.6340.027664CK=CILDTSNPVK
1/y3-2/b1480.1587480.15050.0081107ECK=C
1/y3-2/b4821.3538821.34190.0118460ECK=CILD
1/y3-2/b5922.4014922.37710.0244283ECK=CILDT
1/y3-2/b81220.5291220.5190.0098340ECK=CILDTSNP
1/y3-2/b91319.5981319.5840.0138987ECK=CILDTSNPV
1/y4-2/a1566.2067566.2282−0.0215232NECK=C
1/y4-2/a2679.2908679.3111−0.0203202NECK=CI
1/y4-2/a71209.5241209.5020.0221927NECK=CILDTSN
1/y4-2/b3820.3697820.36670.003154NECK=CIL
1/y4-2/b4935.3967935.39240.0043481NECK=CILD
1/y4-2/b51036.4441036.448−0.0039191NECK=CILDT
1/y4-2/b81334.5721334.57201655NECK=CILDTSNP
1/y4-2/y101579.7461579.6750.07065505NECK=CILDTSNPVK
1/y5-2/b81431.6251431.5990.0256644PNECK=CILDTSNP
1/y5-2/y101676.7991676.7980.001215020PNECK=CILDTSNPVK
1/y6-2/a1776.3435776.33270.0108842IPNECK=C
1/y6-2/a61305.6181305.5390.0798217IPNECK=CILDTS
1/y6-2/b2917.4225917.40440.01811033IPNECK=CI
1/y6-2/b31030.5071030.4130.0941390IPNECK=CIL
1/y6-2/y101789.8831789.8670.01612137IPNECK=CILDTSNPVK
1/z4-2/y101562.721562.6610.05852069NECK=CILDTSNPVK
1/z5-2/y101659.7721659.854−0.08222401PNECK=CILDTSNPVK













Cys74 to Cys85 and Cys90 to Cys100 44 Fragments1/y10-1/a1–2/a21392.591392.5790.0105789NVCIPNECK=C=CI
1/y10-2/b9-1/a52594.1512594.1220.029585NVCIPNECK=CILDTSNPV=CNLGY
1/y11-1/a4-2/a42003.9182003.830.0873148VNNVCIPNECK=CNLG=CILD
1/y11-1/b4-2/b11718.7121718.6860.0265115VNNVCIPNECK=CNLG=C
1/y11-1/b6-2/b62526.0772525.9930.084186VNNVCIPNECK=CNLGYD=CILDTS
1/y12-2/a2-1/a52069.912069.880.0305554MVNNVCIPNECK=CI=CNLGY
1/y12-2/b5-1/a22093.8952093.8330.0625298MVNNVCIPNECK=CILDT=CN
1/y13-1/b1–2/a92492.0752492.0590.0166490DMVNNVCIPNECK=C=CILDTSNPV
1/y13-1/b2-2/a31992.8471992.854−0.0063826DMVNNVCIPNECK=CN=CIL
1/y13-2/b3-1/b42190.9482190.9090.0388313DMVNNVCIPNECK=CIL=CNLG
1/y13-2/b5-1/a22208.9222208.972−0.0498102DMVNNVCIPNECK=CILDT=CN
1/y14-1/a1–2/a82528.0752527.9860.0891572YDMVNNVCIPNECK=C=CILDTSNP
1/y14-2/b9-1/a32882.2662882.240.02541679YDMVNNVCIPNECK=CILDTSNPV=CNL
1/y15-1/a1–2/a92684.1652684.0740.0908230GYDMVNNVCIPNECK=C=CILDTSNPV
1/y15-1/b2-2/b92854.1982854.1660.0322311GYDMVNNVCIPNECK=CN=CILDTSNPV
1/y7-1/b1–2/b21121.4251121.524−0.09883609CIPNECK=C=CI
1/y7-2/a1-1/a21066.3941066.3910.0034286CIPNECK=C=CN
1/y7-2/a1-1/a81744.6991744.779−0.08031284CIPNECK=C=CNLGYDMV
1/y7-2/a2-1/a71758.7151758.6190.0957156CIPNECK=CI=CNLGYDM
1/y7-2/a3-1/a92084.912084.8170.0925164CIPNECK=CIL=CNLGYDMVN
1/y7-2/a5-1/a31621.7211621.6420.0795207CIPNECK=CILDT=CNL
1/y7-2/a9-1/a112911.2922911.2850.0068205CIPNECK=CILDTSNPV=CNLGYDMVNNV
1/y7-2/a9-1/a82584.1382584.0760.062990CIPNECK=CILDTSNPV=CNLGYDMV
1/y7-2/b1-1/a31207.4731207.5−0.027508CIPNECK=C=CNL
1/y7-2/b1-1/a61542.5851542.666−0.0806928CIPNECK=C=CNLGYD
1/y7-2/b2-1/b41405.5741405.5260.0474125CIPNECK=CI=CNLG
1/y7-2/b3-1/b41518.6581518.6430.015197CIPNECK=CIL=CNLG
1/y7-2/b4-1/a102341.9752341.988−0.0132340CIPNECK=CILD=CNLGYDMVNN
1/y7-2/b6-1/a102530.0542529.9850.0696102CIPNECK=CILDTS=CNLGYDMVNN
1/y7-2/b8-1/b31975.8391975.88−0.0417245CIPNECK=CILDTSNP=CNL
1/y8-1/a1–2/a51493.6631493.6290.0336182VCIPNECK=C=CILDT
1/y8-2/b9-1/a32145.9812145.9310.0496164VCIPNECK=CILDTSNPV=CNL
1/y9-1/b1–2/b51663.6951663.6620.0337104NVCIPNECK=C=CILDT
1/y9-2/b1-1/a61755.6961755.718−0.0212658NVCIPNECK=C=CNLGYD
1/y9-2/b1-1/b11221.4531221.521−0.068782NVCIPNECK=C=C
1/y9-2/b8-1/a62496.0672496.0510.0159634NVCIPNECK=CILDTSNP=CNLGYD
1/y9-2/b9-1/a32260.0232259.990.0334668NVCIPNECK=CILDTSNPV=CNL
2/y10-1/y12-1/b22665.1922665.0970.0942258CILDTSNPVK=MVNNVCIPNECK=CN
2/y10-1/y7-1/b103014.3193014.310.00881407CILDTSNPVK=CIPNECK=CNLGYDMVNN
2/y10-1/y7-1/b113113.3883113.393−0.0056120269CILDTSNPVK=CIPNECK=CNLGYDMVNNV
2/y10-1/y7-1/b62556.1242556.0950.02911309CILDTSNPVK=CIPNECK=CNLGYD
2/y10-1/y9-1/a42463.152463.0930.0574609CILDTSNPVK=NVCIPNECK=CNLG
2/y10-1/y9-1/a52626.2142626.1140.0999654CILDTSNPVK=NVCIPNECK=CNLGY
2/y10-1/y9-1/b32434.1242434.0590.0645571CILDTSNPVK=NVCIPNECK=CNL
Fig. 5

Disulfide bonds SS5 and SS6.

Disulfide bonds SS5 and SS6. Disulfide bond SS5 (Cys74-Cys85) and SS6 (Cys90-Cys100) peptides.

Disulfide bonds SS7 and SS8

A total of 39 fragments were observed. Three fragment ions (1/b7-2/b5, 1/y6-2/a4, and 1/y6-2/b4) were specific to the linkage between T12 and T14 and confirmed the Cys95 to Cys113 linkage. Four fragment ions (2/y5-3/b2, 2/y5-3/y3, 2/y6-3/a2, and 2/y6-3/b2) were specific to the linkage between T14 and T16 and confirmed the linkage of Cys115 to Cys129. A further 21 fragment ions were consistent with the linkage of T12, T14, and T16 and remaining 11 fragments consistent with constituent peptides (Table 8, Fig. 6).
Table 8

Disulfide bond SS7 (Cys95-Cys113) and SS8 (Cys115-Cys129) peptides.

AssignmentTheoretical Mass (Da)Observed Mass (Da)Mass Error (Da)Intensity (counts)Identification
39 FragmentsConstituent Peptides 11 Fragments1/a2200.1399200.141−0.0011247QV
1/a3301.1876301.14640.0412100QVT
1/b1129.0664129.06530.001133Q
1/y1147.1133147.1145−0.00121258K
1/y3318.1777318.1789−0.001247NGK
1/y4375.1992375.2−0.000852GNGK
2/a2131.082131.02740.054630T
2/b1102.0555102.0558−0.0003107T
2/b2159.077159.07680.0002140TG
2/y2204.1348204.07050.064330GK
2/y4431.2618431.2624−0.000672NIGK













Cys95 to Cys113 3 Fragments1/b7-2/b51105.4411105.430.0109157QVTCGNG=TGVCS
1/y6-2/a4909.3923909.4301−0.037858TCGNGK=TGVC
1/y6-2/b4937.3871937.3987−0.0115257TCGNGK=TGVC













Cys115 to Cys129 4 Fragments2/y5-3/b2722.2966722.3025−0.005945CNIGK=CS
2/y5-3/y3868.4021868.35440.0477103CNIGK=CSK
2/y6-3/a2781.3337781.4172−0.083521SCNIGK=CS
2/y6-3/b2809.3286809.32590.002790SCNIGK=CS













T12, T14, and T16 linkage 21 Fragments1/a4-2/a6-3/a1997.3728997.3982−0.025439QVTC=TGVCSC=C
1/a4-2/a6-3/a21084.4051084.434−0.0294146QVTC=TGVCSC=CS
1/a4-2/a8-3/b21339.5271339.585−0.0582210QVTC=TGVCSCNI=CS
1/a4-2/y8-3/y31558.6851558.640.0449111QVTC=VCSCNIGK=CSK
1/a5-2/a6-3/a11054.3941054.432−0.037660QVTCG=TGVCSC=C
1/a6-2/y8-3/y31729.7491729.660.08951QVTCGN=VCSCNIGK=CSK
1/a7-2/a7-3/b11367.4971367.4720.024433QVTCGNG=TGVCSCN=C
1/b5-2/a9-3/b21481.5651481.4930.071335QVTCG=TGVCSCNIG=CS
1/b6-2/a7-3/b11338.471338.53−0.059672QVTCGN=TGVCSCN=C
1/b6-2/b9-3/y31769.7081769.6510.05661155QVTCGN=TGVCSCNIG=CSK
1/y5-2/b6-3/y31360.5121360.561−0.0487249CGNGK=TGVCSC=CSK
1/y5-2/b9-3/b11411.5231411.599−0.0762348CGNGK=TGVCSCNIG=C
1/y5-2/y8-3/a11371.5641371.5290.035254CGNGK=VCSCNIGK=C
1/y5-2/y8-3/b21486.5911486.645−0.0536270CGNGK=VCSCNIGK=CS
1/y6-2/y10-3/y31891.8141891.7610.0521105TCGNGK=TGVCSCNIGK=CSK
1/y7-2/b8-3/b21641.6491641.6310.018878VTCGNGK=TGVCSCNI=CS
1/y7-2/y10-3/y31990.8821990.7980.0837124VTCGNGK=TGVCSCNIGK=CSK
1/y7-2/y8-3/b21686.7071686.75−0.04246211VTCGNGK=VCSCNIGK=CS
1/y8-2/b8-3/b11682.6761682.723−0.046632QVTCGNGK=TGVCSCNI=C
1/y8-2/b9-3/y31972.8351972.8190.0156407QVTCGNGK=TGVCSCNIG=CSK
1/y8-2/y10-3/y32118.942118.9320.00854955QVTCGNGK=TGVCSCNIGK=CSK
Fig. 6

Disulfide bonds SS7 and SS8.

Disulfide bonds SS7 and SS8. Disulfide bond SS7 (Cys95-Cys113) and SS8 (Cys115-Cys129) peptides.

Disulfide bond SS9

A total of 35 fragment ions were observed with 25 fragment ions of this peptide consistent with the linkage of T18 to T20 through Cys137 and Cys148. Ten fragments were consistent with constituent peptides (Table 9, Fig. 7).
Table 9

Disulfide bond SS9 (Cys137-Cys148) peptides.

AssignmentTheoretical Mass (Da)Observed Mass (Da)Mass Error (Da)Intensity (counts)Identification
35 Fragmentsconstituent peptides 10 Fragments1/y2260.1974260.2041−0.0067262LK
1/y3347.2294347.2299−0.0005129SLK
2/a1102.0555102.0559−0.0004754E
2/a2216.0984216.1371−0.038765EN
2/a4446.1887446.2249−0.036220ENET
2/b1130.0504130.04690.003517E
2/b2244.0933244.09290.0004367EN
2/b3373.1359373.1376−0.0017171ENE
2/b4474.1836474.1958−0.012288ENET
2/y1147.1133147.113301131K













Cys137 and Cys148 25 Fragments1/a1–2/y2323.1212323.10080.020415C=CK
1/a1–2/y4553.2114553.20580.005653C=ETCK
1/a1–2/y5667.2544667.2748−0.020459C=NETCK
1/a2-2/y2410.1532410.1623−0.009136CS=CK
1/a2-2/y4640.2435640.2652−0.021734CS=ETCK
1/a3-2/y3624.2849624.2893−0.004427CSL=CKT
1/a3-2/y5867.3705867.35080.019789CSL=NETCK
1/b1–2/a5650.1914650.2377−0.0463130C=ENETC
1/b2-2/a5737.2234737.2545−0.0311139CS=ENETC
1/b1–2/y2351.1161351.1717−0.0556207C=CK
1/b1–2/y3452.1638452.1826−0.018823C=CKT
1/b2-2/y2438.1481438.1512−0.0031139CS=CK
1/b2-2/y3539.1958539.2008−0.005166CS=CKT
1/b2-2/y5782.2813782.3434−0.0621180CS=NETCK
1/b3-2/y2551.2322551.2324−0.0002219CSL=CK
1/y4-2/y2697.3377697.33290.0048465CSLK=CK
1/y4-2/a5996.413996.40690.0061130CSLK=ENETC
1/y4-2/b51024.4081024.4126−0.0046502CSLK=ENETC
1/y4-2/y3798.3854798.38020.0052553CSLK=TCK
1/y4-2/y4927.428927.42160.0064223CSLK=ETCK
1/y4-2/y51041.47091041.43770.033294CSLK=NETCK
1/y4-2/y61170.51341170.5110.002425002CSLK=ENETCK
1/a1–2/y6796.2969796.3787−0.0818646C=ENETCK
1/b2-2/y6911.3239911.29630.0276155CS=ENETCK
1/y4-2/z3781.3588781.32290.035977CSLK=TCK
Fig. 7

Disulfide bond SS9.

Disulfide bond SS9. Disulfide bond SS9 (Cys137-Cys148) peptides.

Disulfide bonds SS10 and SS11

A total of 65 fragments were observed. Eleven fragment ions were specific to the linkage of T19 and T22, and confirmed the linkage of Cys141 to Cys157. Three fragment ions were specific to the linkage between peptides T22 and T23 and confirmed the linkage of Cys159 to Cys172. Thirty-two fragment ions were consistent with the linkage of T12, T14, and T16 and an additional 19 fragments were consistent with constituent peptides (Table 10, Fig. 8).
Table 10

Disulfide bond SS10 (Cys141-Cys157) and SS11 (Cys159-Cys172) peptides.

AssignmentTheoretical Mass (Da)Observed Mass (Da)Mass Error (Da)Intensity (counts)Identification
65 FragmentsConstituent Peptides 19 fragments1/y1147.1133147.11260.00071153K
1/y2260.1974260.2021−0.0047167LK
3/a2145.0613145.0646−0.0033400DG
3/a5518.2979518.2960.0018109DGFII
3/a8890.426890.35210.073984DGFIIDQE
3/a9977.458977.36660.0914120DGFIIDQES
3/b1116.0348116.0532−0.018428D
3/b2173.0562173.05610.0001645DG
3/b3320.1246320.1256−0.0016533DGF
3/b4433.2087433.20640.00231906DGFI
3/b5546.2927546.28840.0043325DGFII
3/b6661.3197661.30640.0133295DGFIID
3/b91005.4531005.3830.0699237DGFIIDQES
3/y1156.0773156.07690.00044578H
3/y2293.1362293.1368−0.00061684HH
3/y3430.1951430.19130.00381430HHH
3/y4567.254567.2552−0.00121460HHHH
3/y5704.3129704.30940.00351025HHHHH
3/y7942.4196942.40530.0143568THHHHHH













Cys141 to Cys157 11 Fragments1/a1–2/a1149.0207149.1165−0.095816C=C
1/a2-2/a1262.1048262.1324−0.027639CL=C
1/a2-2/a2377.1317377.1469−0.0152258CL=CD
1/a2-2/b1290.0997290.1243−0.024645CL=C
1/a2-2/b2405.1266405.12620.0005230CL=CD
1/y3-2/a2551.2322551.20630.025962CLK=CD
1/b1–2/a2292.0426292.03120.011482C=CD
1/b1–2/b1205.0106205.0966−0.086154C=C
1/b2-2/b1318.0946318.08720.007436CL=C
1/y3-2/b1464.2001464.214−0.013941CLK=C
1/y3-2/b2579.2271579.2289−0.00181027CLK=CD















Cys159 to Cys172 3 fragments2/y2–3/a121527.6821527.6530.0291242CK=DGFIIDQESSIC
2/y2–3/y192497.0892497.0660.0227994CK=DGFIIDQESSICTHHHHHH
2/y3-3/a121642.7091642.6330.076369DCK=DGFIIDQESSIC















T12, T14, and T16 Linkage 32 Fragments1/a1–2/a3-3/a162156.84112156.8150.026637C=CDC=DGFIIDQESSICTHHH
1/a1–2/a3-3/y101609.57821609.5590.0193132C=CDC=SICTHHHHHH
1/a1–2/a3-3/y142068.73832068.799−0.060874C=CDC=DQESSICTHHHHHH
1/a1–2/y4-3/y182673.09672673.159−0.062362C=CDCK=GFIIDQESSICTHHHHHH
1/a1–2/y4-3/y81583.56251583.616−0.0533363C=CDCK=CTHHHHHH
1/a2-2/a3-3/a162269.9252269.920.0051921CL=CDC=DGFIIDQESSICTHHH
1/a2-2/a3-3/a182544.0432544.0350.007658CL=CDC=DGFIIDQESSICTHHHHH
1/a2-2/y4-3/a152306.96662306.9040.0623147CL=CDCK=DGFIIDQESSICTHH
1/b1–2/b3-3/a121700.60641700.657−0.0505100C=CDC=DGFIIDQESSIC
1/b1–2/b3-3/y142124.72832124.776−0.047672C=CDC=DQESSICTHHHHHH
1/b1–2/y4-3/a142084.81862084.862−0.043785C=CDCK=DGFIIDQESSICTH
1/b1–2/y4-3/y172644.07032644.117−0.04712248C=CDCK=FIIDQESSICTHHHHHH
1/b2-2/b3-3/a121813.69061813.709−0.018163CL=CDC=DGFIIDQESSIC
1/b2-2/b3-3/b131942.73321942.833−0.099795CL=CDC=DGFIIDQESSICT
1/b2-2/b3-3/y81578.5361578.608−0.0721228CL=CDC=CTHHHHHH
1/b2-2/b3-3/y91691.621691.693−0.0729105CL=CDC=ICTHHHHHH
1/b2-2/y4-3/a142197.90262197.909−0.0066671CL=CDCK=DGFIIDQESSICTH
1/b2-2/y4-3/b172637.07422637.0170.0576233CL=CDCK=DGFIIDQESSICTHHHH
1/b2-2/y4-3/y101924.75761924.81−0.052484CL=CDCK=SICTHHHHHH
1/b2-2/y4-3/y162610.08592610.088−0.0017407CL=CDCK=IIDQESSICTHHHHHH
1/y3-2/y4-3/a182892.24392892.1640.0803403CLK=CDCK=DGFIIDQESSICTHHHHH
1/y3-2/y4-3/b142372.00322372.038−0.0352341CLK=CDCK=DGFIIDQESSICTH
1/y3-2/y4-3/b182920.23882920.248−0.00881160CLK=CDCK=GFIIDQESSICTHHHHHH
1/y3-2/y4-3/y112157.89532157.8930.0024922CLK=CDCK=SSICTHHHHHH
1/y3-2/y4-3/y132414.99632415.007−0.011897CLK=CDCK=QESSICTHHHHHH
1/y3-2/y4-3/y142530.02322530.028−0.00462415CLK=CDCK=DQESSICTHHHHHH
1/y3-2/y4-3/y162756.19142756.2−0.00881421CLK=CDCK=IIDQESSICTHHHHHH
1/y3-2/y4-3/y182960.28122960.2380.043911CLK=CDCK=GFIIDQESSICTHHHHHH
1/y3-2/y4-3/y193075.30813075.3080.000542471CLK=CDCK=DGFIIDQESSICTHHHHHH
1/y3-2/y4-3/y81870.74711870.768−0.0212131CLK=CDCK=CTHHHHHH
1/y3-2/y4-3/z142512.99682513.063−0.0657102CLK=CDCK=DQESSICTHHHHHH
1/y3-2/y4-3/z81853.72051853.793−0.072466CLK=CDCK=CTHHHHHH
Fig. 8

Disulfide bonds SS10 and SS11.

Disulfide bonds SS10 and SS11. Disulfide bond SS10 (Cys141-Cys157) and SS11 (Cys159-Cys172) peptides.

Experimental design, materials and methods

Sample preparation

Baculovirus Pfs25 [2] was denatured and digested as described in [1].

Chromatography

Digested peptides were separated with a 2695 Separations Module (Waters Corporation; Milford MA) and a 2489 UV/Vis Detector (Waters Corporation; Milford, MA) set at 214 nm. An XBridge (Waters Corporation; Milford, MA) BEH 300 C18 (2.1×250 mm, 5 µm) was used at a column temperature of 37 °C and gradient with 0.1% Triflouroacetic acid (TFA) in purified water (Mobile Phase A) and 0.1% TFA in acetonitrile (Mobile Phase B) as described in [1].

Mass spectrometry

MS analysis was done with a QTOF Premier mass spectrometer (Waters Corporation; Milford, MA) equipped with an electrospray source as described in [1]. MS data was acquired in MSE mode using MassLynx v4.1 (Waters Corporation; Milford, MA). RAW MS files have been deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982.

Analysis of mass spectra

The mass spectral data was analyzed using BiopharmaLynx 1.3 (Waters Corporation; Milford, MA) as described in [1].
Subject areaChemistry, Biology,
More specific subject areaDisulfide bond analysis by liquid chromatography and mass spectrometric analysis
Type of dataTables, figures
How data was acquiredData was generated using liquid chromatography (Waters 2695 Separations Module and Waters 2489 UV/Vis Detector) and mass spectroscopy (Waters QTOF Premier mass spectrometer)
Data formatCollated data from analysis with Waters BiopharmaLynx 1.3 and MassLynx
Experimental factorsRecombinant Pfs25 digested with 20 µg of trypsin/Lys-C at 37 °C overnight and subsequent further digestion by additional 20 µg of trypsin/Lys-C for 3–4 hours at 37 °C
Experimental featuresIdentification of the proper pairing of 11 disulfide bonds in Pfs25 through digestion of peptides and LC-MS/MS
Data source locationMass spectrometry data acquired in Middleton, WI, USA
Data accessibilityData is provided within this article and RAW MS files have been deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982 (ftp://massive.ucsd.edu/MSV000081982). MassIVE is a member of the ProteomeXchange Consortium
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Authors:  Nicholas A Pullen; Emma Bertran
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Journal:  Sci Rep       Date:  2020-01-15       Impact factor: 4.379
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