RESUMO
Bottom-up proteomic experiments often require selective conjugation or labeling of the N- and/or C-termini of peptides resulting from proteolytic digestion. For example, techniques based on surface fluorescence imaging are emerging as a promising route to high-throughput protein sequencing but require the generation of peptide surface arrays immobilized through single C-terminal point attachment while leaving the N-terminus free. While several robust approaches are available for selective N-terminal conjugation, it has proven to be much more challenging to implement methods for selective labeling or conjugation of the C-termini that can discriminate between the C-terminal carboxyl group and other carboxyl groups on aspartate and glutamate residues. Further, many approaches based on conjugation through amide bond formation require protection of the N-terminus to avoid unwanted cross-linking reactions. To overcome these challenges, herein, we describe a new strategy for single-point selective immobilization of peptides generated by protease digestion via the C-terminus. The method involves immobilization of peptides via lysine amino acids which are found naturally at the C-terminal end of cleaved peptides from digestions of certain serine endoproteinases, like LysC. This lysine and the N-terminus, the sole two primary amines in the peptide fragments, are chemically reacted with a custom phenyl isothiocyanate (EPITC) that contains an alkyne handle. Subsequent exposure of the double-modified peptides to acid selectively cleaves the N-terminal amino acid, while the modified C-terminus lysine remains unchanged. The alkyne-modified peptides with free N-termini can then be immobilized on an azide surface through standard click chemistry. Using this general approach, surface functionalization is demonstrated using a combination of X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM).
Assuntos
Peptídeo Hidrolases , Proteômica , Alcinos , Lisina/química , Peptídeos/química , Proteômica/métodosRESUMO
El ambiente en que vivimos. Aspectos generales. La problemática ambiental. Humanidad y ambiente. La acción humana. Que podemos hacer
Assuntos
Meio Ambiente , Desenvolvimento SustentávelRESUMO
El objetivo es evaluar el impacto y la eficacia d ela campaña educativa de prevención del SIDA en las poblaciones con actividades de alto riesgo: mujeres que ejercen la prostitución,varones con conducta homosexual y presidiarios en las ciudades de La Paz y Santa Cruz.
