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主页蛋白质标记和修饰肽修饰:N-末端、内部、和C-末端

肽修饰:N-末端、内部、和C-末端


1.0 N-末端修饰

1.1 乙酰化

该修饰去除肽的N-末端上的正电荷,从而模拟天然蛋白质。在某些情况下,它通过阻止N-末端降解来增加肽的稳定性1-2

乙酰化

参考文献

1.
Arnesen T. Towards a Functional Understanding of Protein N-Terminal Acetylation. PLoS Biol. 9(5):e1001074. https://doi.org/10.1371/journal.pbio.1001074
2.
Wallace RJ. 1992. Acetylation of peptides inhibits their degradation by rumen micro-organisms. Br J Nutr. 68(2):365-372. https://doi.org/10.1079/bjn19920095

1.2  生物素

生物素对链霉亲和素及亲和素具有非常强的亲和力。生物素标记的肽通常用于免疫测定1、组织细胞化学2、以及基于荧光的流式细胞计数3

生物素

参考文献

1.
Sélo I, Négroni L, Créminon C, Grassi J, Wal J. 1996. Preferential labeling of ?-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays. Journal of Immunological Methods. 199(2):127-138. https://doi.org/10.1016/s0022-1759(96)00173-1
2.
HOWL J, WANG X, KIRK CJ, WHEATLEY M. 1993. Fluorescent and biotinylated linear peptides as selective bifunctional ligands for the V1a vasopressin receptor. Eur J Biochem. 213(2):711-719. https://doi.org/10.1111/j.1432-1033.1993.tb17811.x
3.
Buranda, et. al. 1991. Peptide, antibodies and FRET on beads in flow cytometry: a model system using fluoresceinated and biotinylated β-endorphin. Cytometry. 3721-31.

1.3  丹磺酰

丹磺酰基标记的肽用于荧光测定。

丹磺酰

参考文献

1.
Glukhov E, Stark M, Burrows LL, Deber CM. 2005. Basis for Selectivity of Cationic Antimicrobial Peptides for BacterialVersusMammalian Membranes. J. Biol. Chem.. 280(40):33960-33967. https://doi.org/10.1074/jbc.m507042200
2.
Matsuzaki K, Mitani Y, Akada K, Murase O, Yoneyama S, Zasloff M, Miyajima K. 1998. Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa?. Biochemistry. 37(43):15144-15153. https://doi.org/10.1021/bi9811617
3.
Pecht I, Maron E, Arnon R, Sela M. 1971. Specific Excitation Energy Transfer from Antibodies to Dansyl-Labeled Antigen.Studies with the "Loop" Peptide of Hen Egg-White Lysozyme. Eur J Biochem. 19(3):368-371. https://doi.org/10.1111/j.1432-1033.1971.tb01325.x

1.4   2,4-二硝基苯基

2,4-DNP用作(7-甲氧基香豆素-4-基)乙酰基(MCA)猝灭剂,有时也作为色氨酸的猝灭剂。该修饰可以附着在肽的N-末端,或通过赖氨酸侧链作为内部修饰。

二硝基苯基

参考文献

1.
Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F, et al. 2002. Hydrolysis of Biological Peptides by Human Angiotensin-converting Enzyme-related Carboxypeptidase. J. Biol. Chem.. 277(17):14838-14843. https://doi.org/10.1074/jbc.m200581200
2.
Knight C, Willenbrock F, Murphy G. 1992. A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. 296(3):263-266. https://doi.org/10.1016/0014-5793(92)80300-6

1.5   荧光素

荧光素标记的肽具有许多基于荧光素的生物分子应用,包括蛋白质-蛋白质相互作用、流式细胞计数和定位研究1-3

荧光素

参考文献

1.
Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV, Lebleu B. 2003. Cell-penetrating Peptides. J. Biol. Chem.. 278(1):585-590. https://doi.org/10.1074/jbc.m209548200
2.
Farley RA, Tran CM, Carilli CT, Hawke D, Shively JE. 1984. The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na,K)-ATPase. J Biol Chem. 259(15):9532-5.
3.
Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, Sugiura Y. 2001. Arginine-rich Peptides. J. Biol. Chem.. 276(8):5836-5840. https://doi.org/10.1074/jbc.m007540200
4.
Foerg C, Weller KM, Rechsteiner H, Nielsen HM, Fernández-Carneado J, Brunisholz R, Giralt E, Merkle HP. 2008. Metabolic Cleavage and Translocation Efficiency of Selected Cell Penetrating Peptides: A Comparative Study with Epithelial Cell Cultures. AAPS J. 10(2):349-359. https://doi.org/10.1208/s12248-008-9029-4

1.6  7-甲氧基香豆素乙酸(Mca)

7-甲氧基香豆素标记的肽在蛋白质-蛋白质相互作用和定位研究中有应用1-3

7-甲氧基香豆素乙酸(Mca)

参考文献

1.
Vidal, et. al. 1996. Solid-Phase Synthesis and Cellular Localization of a C-and/or N-terminal Labeled Peptide. Journal of Peptide Science. 2125-133.
2.
Yandek LE, Pokorny A, Florén A, Knoelke K, Langel &, Almeida PF. 2007. Mechanism of the Cell-Penetrating Peptide Transportan 10 Permeation of Lipid Bilayers. Biophysical Journal. 92(7):2434-2444. https://doi.org/10.1529/biophysj.106.100198

1.7  棕榈酸

棕榈酸是16-碳脂肪酸,其与肽缀合以增加其细胞渗透性,并有助于肽与细胞膜的结合1-2

棕榈酸

参考文献

1.
Avrahami D, Shai Y. 2004. A New Group of Antifungal and Antibacterial Lipopeptides Derived from Non-membrane Active Peptides Conjugated to Palmitic Acid. J. Biol. Chem.. 279(13):12277-12285. https://doi.org/10.1074/jbc.m312260200
2.
Buss JE, Sefton BM. 1986. Direct identification of palmitic acid as the lipid attached to p21ras.. Mol.Cell.Biol.. 6(1):116-122. https://doi.org/10.1128/mcb.6.1.116

2.0 内部修饰

2.1  环化(二硫键)

该过程涉及在两个半胱氨酸残基之间形成二硫键。肽可以被环化以稳定肽构象,增加生物活性和酶稳定性1-2

参考文献

1.
Góngora-Benítez M, Tulla-Puche J, Albericio F. 2014. Multifaceted Roles of Disulfide Bonds.Peptides as Therapeutics. Chem. Rev.. 114(2):901-926. https://doi.org/10.1021/cr400031z
2.
Schmelz EA, Huffaker A, Carroll MJ, Alborn HT, Ali JG, Teal PE. 2012. An Amino Acid Substitution Inhibits Specialist Herbivore Production of an Antagonist Effector and Recovers Insect-Induced Plant Defenses. Plant Physiol.. 160(3):1468-1478. https://doi.org/10.1104/pp.112.201061

2.2 半胱氨酸脲基甲基化(CAM)

脲基甲基化(CAM)是一种故意的翻译后修饰,通过与碘乙酰胺反应引入到半胱氨酸残基。具有这种修饰的肽主要用于肽质量指纹图谱中,以鉴定和表征蛋白质1。在其他测定中,该过程用于阻断半胱氨酸的氧化2

半胱氨酸脲基甲基化 (CAM)

参考文献

1.
Wilkins MR, Appel RD, Williams KL, Hochstrasser DF. 2007. Proteome Research. https://doi.org/10.1007/978-3-540-72910-5
2.
Rombouts I, Lagrain B, Brunnbauer M, Delcour JA, Koehler P. 2013. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry. Sci Rep. 3(1): https://doi.org/10.1038/srep02279

2.3 同位素标记的氨基酸

AQUA肽是18O、13C、和/或14N中富含的氨基酸的合成肽。它们在化学、物理性质以及生物活性方面与它们的天然肽相似1。这些肽的主要应用是研究蛋白质相互作用、蛋白质、翻译后修饰(如泛素化和磷酸化)2-5

参考文献

1.
Kettenbach AN, Rush J, Gerber SA. 2011. Absolute quantification of protein and post-translational modification abundance with stable isotope?labeled synthetic peptides. Nat Protoc. 6(2):175-186. https://doi.org/10.1038/nprot.2010.196
2.
Li H, Xu C, Blais S, Wan Q, Zhang H, Landry SJ, Hioe CE. 2009. Proximal Glycans Outside of the Epitopes Regulate the Presentation of HIV-1 Envelope gp120 Helper Epitopes. J Immunol. 182(10):6369-6378. https://doi.org/10.4049/jimmunol.0804287
3.
Le Bihan T, Grima R, Martin S, Forster T, Le Bihan Y. 2010. Quantitative analysis of low-abundance peptides in HeLa cell cytoplasm by targeted liquid chromatography/mass spectrometry and stable isotope dilution: emphasising the distinction between peptide detection and peptide identification. Rapid Commun.Mass Spectrom.. 24(7):1093-1104. https://doi.org/10.1002/rcm.4487
4.
Santhoshkumar P, Raju M, Sharma KK. ?A-Crystallin Peptide 66SDRDKFVIFLDVKHF80 Accumulating in Aging Lens Impairs the Function of ?-Crystallin and Induces Lens Protein Aggregation. PLoS ONE. 6(4):e19291. https://doi.org/10.1371/journal.pone.0019291
5.
Sato Y, Miyashita A, Iwatsubo T, Usui T. 2012. Simultaneous Absolute Protein Quantification of Carboxylesterases 1 and 2 in Human Liver Tissue Fractions using Liquid Chromatography-Tandem Mass Spectrometry. Drug Metab Dispos. 40(7):1389-1396. https://doi.org/10.1124/dmd.112.045054
6.
Brun V, Masselon C, Garin J, Dupuis A. 2009. Isotope dilution strategies for absolute quantitative proteomics. Journal of Proteomics. 72(5):740-749. https://doi.org/10.1016/j.jprot.2009.03.007

2.4 磷酸化

可以对Tyr、Ser和Thr残基进行磷酸化,作为肽上的翻译后修饰(PTM)。磷酸化肽可用于许多细胞过程,如基因表达、蛋白质-蛋白质相互作用以及植物和动物中的信号传导1,2

磷酸化

参考文献

1.
Guenther JF, Chanmanivone N, Galetovic MP, Wallace IS, Cobb JA, Roberts DM. 2003. Phosphorylation of Soybean Nodulin 26 on Serine 262 Enhances Water Permeability and Is Regulated Developmentally and by Osmotic Signals. Plant Cell. 15(4):981-991. https://doi.org/10.1105/tpc.009787

2.5 间隔

间隔物用于在肽和货物之间产生距离,以减少肽结合位点的空间位阻。在这种情况下,货物可以是药物、染料、标记。

2.5.1 聚乙二醇化(PEG化)

聚(乙二醇)与肽的连接称为PEG化。短的双功能PEG(聚(乙二醇))可用作肽与其他分子的生物共轭中的间隔物。PEG生物共轭也已用于改善蛋白水解稳定性、肽的生物分布和溶解度1-2

聚乙二醇化

参考文献

1.
Sullivan TP, van Poll ML, Dankers PYW, Huck WTS. 2004. Forced Peptide Synthesis in Nanoscale Confinement under Elastomeric Stamps. Angew.Chem.. 116(32):4286-4289. https://doi.org/10.1002/ange.200460271
2.
Veronese FM. 2001. Peptide and protein PEGylation. Biomaterials. 22(5):405-417. https://doi.org/10.1016/s0142-9612(00)00193-9

2.5.2 氨基己酸

氨基己酸是一种疏水间隔物,附着在它上面的分子、荧光团、标记、或任何生物分子可以附着到肽上1-2

氨基己酸

参考文献

1.
Mitchell D, Steinman L, Kim D, Fathman C, Rothbard J. 2000. Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res. 56(5):318-325. https://doi.org/10.1034/j.1399-3011.2000.00723.x

3.0 C-末端修饰

3.1  酰胺(酰胺化)

肽的C-末端合成为酰胺,以中和由C-末端COOH产生的负电荷。添加这种修饰是为了防止酶降解,以模拟天然蛋白质,并且在某些情况下除去可能干扰测定的结合在肽C-末端的氢键。1

参考文献

1.
Kim K, Seong BL. 2001. Peptide amidation: Production of peptide hormonesin vivo andin vitro. Biotechnol.Bioprocess Eng.. 6(4):244-251. https://doi.org/10.1007/bf02931985