High-resolution peptide separations using nano-LC at ultra-high pressure.
J Sep Sci
; 36(7): 1192-9, 2013 Apr.
Article
em En
| MEDLINE
| ID: mdl-23457143
ABSTRACT
We report on the optimization of nano-LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 µm id capillary columns packed with 2, 3, and 5 µm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 µm particle-packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic-performance limit, i.e. using 2-µm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run-to-run retention time stability (RSD values below 0.18%) was demonstrated applying ultra-high pressure conditions.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Cromatografia Líquida de Alta Pressão
/
Nanotecnologia
Idioma:
En
Revista:
J Sep Sci
Ano de publicação:
2013
Tipo de documento:
Article