US 2021/0190794 A1 · COMPOSITIONS AND METHODS FOR PROTEIN DETECTION

Abstract

The invention relates generally to peptide biomarkers with specific ionization characteristics to directly quantify one or more transgenic target proteins in biological samples, including transgenic plant samples, by liquid chromatography coupled tandem mass spectrometry multiple reaction monitoring (MRM). The peptide biomarkers in combination with MRM-based methods may be used to quantify a single transgenic target protein or multiple transgenic target proteins within a stacked transgenic crop, such as maize, utilizing selected peptide biomarkers either alone or in combination. The present disclosure allows for broad based, reliable quantitation in different biological matrices, including plant matrices. The peptide biomarkers of the invention can further be used as trait biomarkers to support identification and/or selection of specific transgenic Events. Also provided are different peptide biomarker combinations that can be used to perform the methods of the invention.

A labeled surrogate peptide that functions in a mass spectrometry assay to selectively detect or quantitate a target transgenic protein selected from the group consisting of a Cry1Ab protein, an eCry3.1Ab protein, a mCry3A protein, a Vip3 protein, a double mutant 5-enolpyruvylshikimate-3-phosphate synthase (dmEPSPS) protein, a phosphinothricin acetyltransferase (PAT) protein and a phosphomannose isomerase (PMI) protein in a mixture of transgenic proteins and non-transgenic proteins in one or more biological samples from one or more transgenic plants, the surrogate peptide comprising a label and an amino acid sequence selected from the group consisting of

GSAQGIEGSIR,
(SEQ ID NO:
1)
IVAQLGQGVYR,
(SEQ ID NO:
2)
TLSSTLYR,
(SEQ ID NO:
3)
DVSVFGQR,
(SEQ ID NO:
4)
TYPIR,
(SEQ ID NO:
5)
TVSQLTR,
(SEQ ID NO:
6)
WYNTGLER,
(SEQ ID NO:
7)
EWEADPTNPALR,
(SEQ ID NO:
8)
VWGPDSR,
(SEQ ID NO:
9)
APMFSWIHR,
(SEQ ID NO:
10)
WGFDAATINSR,
(SEQ ID NO:
11)
NQAISR,
(SEQ ID NO:
12)
IEEFAR,
(SEQ ID NO:
13)
SGFSNSSVSIIR,
(SEQ ID NO:
14)
LSHVSMFR,
(SEQ ID NO:
15)
EIYTNPVLENFDGSFR,
(SEQ ID NO:
16)
LEGLSNLYQIYAESFR,
(SEQ ID NO:
17)
YNQFR,
(SEQ ID NO:
18)
YNDLTR,
(SEQ ID NO:
19)
SPHLMDILNSITIYTDAHR,
(SEQ ID NO:
20)
SAEFNNIIPSSQITQIPLTK,
(SEQ ID NO:
21)
QGFSHR,
(SEQ ID NO:
22)
MDNNPNINECIPYNCLSNPEVEVLGGER,
(SEQ ID NO:
23)
ELTLTVLDIVSLFPNYDSR,
(SEQ ID NO:
24)
RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR,
(SEQ ID NO:
25)
SGTVDSLDEIPPQNNNVPPR,
(SEQ ID NO:
26)
TDVTDYHIDQV,
(SEQ ID NO:
27)
AVNELFTSSNQIGLK,
(SEQ ID NO:
28)
ITQLPLTK,
(SEQ ID NO:
29)
GLDSSTTK,
(SEQ ID NO:
30)
QCAGIRPYDGR,
(SEQ ID NO:
31)
IEFVPAEVTFEAEYDLER,
(SEQ ID NO:
32)
ITQLPLVK,
(SEQ ID NO:
33)
MTADNNTEALDSSTTK,
(SEQ ID NO:
34)
VYIDK,
(SEQ ID NO:
35)
DGGISQFIGDK,
(SEQ ID NO:
36)
LITLTCK,
(SEQ ID NO:
37)
ELLLATDLSNK,
(SEQ ID NO:
38)
FEELTFATETSSK,
(SEQ ID NO:
39)
EVLFEK,
(SEQ ID NO:
40)
TASELITK,
(SEQ ID NO:
41)
DVSEMFTTK,
(SEQ ID NO:
42)
LLGLADIDYTSIMNEHLNK,
(SEQ ID NO:
43)
IDFTK,
(SEQ ID NO:
44)
TDTGGDLTLDEILK,
(SEQ ID NO:
45)
DIMNMIFK,
(SEQ ID NO:
46)
ALYVHK,
(SEQ ID NO:
47)
VNILPTLSNTFSNPNYAK,
(SEQ ID NO:
48)
ITSMLSDVIK,
(SEQ ID NO:
49)
QNLQLDSFSTYR,
(SEQ ID NO:
50)
DSLSEVIYGDMDK,
(SEQ ID NO:
51)
MIVEAKPGHALIGFEISNDSITVLK,
(SEQ ID NO:
52)
VYFSVSGDANVR,
(SEQ ID NO:
53)
NQQLLNDISGK,
(SEQ ID NO:
54)
VESSEAEYR,
(SEQ ID NO:
55)
YMSGAK,
(SEQ ID NO:
56)
DGSPADILDELTELTELAK,
(SEQ ID NO:
57)
VYEAK,
(SEQ ID NO:
58)
LDAINTMLR,
(SEQ ID NO:
59)
GKPSIHLK,
(SEQ ID NO:
60)
DENTGYIHYEDTNNNLEDYQTINK,
(SEQ ID NO:
61)
DNFYIELSQGNNLYGGPIVHFYDVSIK,
(SEQ ID NO:
62)
LLCPDQSEQIYYTNNIVFPNEYVITK,
(SEQ ID NO:
63)
SQNGDEAWGDNFIILEISPSEK,
(SEQ ID NO:
64)
NAYVDHTGGVNGTK,
(SEQ ID NO:
65)
LDGVNGSLNDLIAQGNLNTELSK,
(SEQ ID NO:
66)
IANEQNQVLNDVNNK,
(SEQ ID NO:
67)
YEVTANFYDSSTGEIDLNK,
(SEQ ID NO:
68)
QNYALSLQIEYLSK,
(SEQ ID NO:
69)
QLQEISDK,
(SEQ ID NO:
70)
LLSPELINTNNWTSTGSTNISGNTLTLYQGGR,
(SEQ ID NO:
71)
YVNEK,
(SEQ ID NO:
72)
QNYQVDK,
(SEQ ID NO:
73)
MAGAEEIVLQPIK,
(SEQ ID NO:
74)
FPVEDAK,
(SEQ ID NO:
75)
EISGTVK,
(SEQ ID NO:
76)
ILLLAALSEGTTVVDNLLNSEDVHYMLGALR,
(SEQ ID NO:
77)
DFELPAPPRPVRPVTQI,
(SEQ ID NO:
78)
LGLGSTLYTHLLK,
(SEQ ID NO:
79)
MSPER,
(SEQ ID NO:
80)
HGGWHDVGFWQR,
(SEQ ID NO:
81)
NAYDWTVESTVYVSHR,
(SEQ ID NO:
82)
TEPQTPQEWIDDLER,
(SEQ ID NO:
83)
AAGYK,
(SEQ ID NO:
84)
YPWLVAEVEGVVAGIAYAGPWK,
(SEQ ID NO:
85)
RPVEIRPATAADMAAVCDIVNHYIETSTVNFR,
(SEQ ID NO:
86)
ENAAGIPMDAAER,
(SEQ ID NO:
87)
ALAILK,
(SEQ ID NO:
88)
SALDSQQGEPWQTIR,
(SEQ ID NO:
89)
GSQQLQLKPGESAFIAANESPVTVK,
(SEQ ID NO:
90)
FEAKPANQLLTQPVK,
(SEQ ID NO:
91)
STLLGEAVAK,
(SEQ ID NO:
92)
LINSVQNYAWGSK,
(SEQ ID NO:
93)
HNSEIGFAK,
(SEQ ID NO:
94)
VLCAAQPLSIQVHPNK,
(SEQ ID NO:
95)
TALTELYGMENPSSQPMAELWMGAHPK,
(SEQ ID NO:
96)
LSELFASLLNMQGEEK,
(SEQ ID NO:
97)
and
QGAELDFPIPVDDFAFSLHDLSDK,
(SEQ ID NO:
98)
LQSGASVVAGPR,
(SEQ ID NO:
252)
FTTGTDLK
(SEQ ID NO:
255)
and
SLTAAVTAAGGNATYVLDGVPR.
(SEQ ID NO:
257)

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Compositions and methods for protein detection

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Compositions and methods for protein detection

Life of this patent

Abstract

Claims36