Agilent Tools considerations to increase resolution of complex proteome samples by two-dimensional offline LC/MS
Update: 30 September, 2023
This Application Note describes a method for increasing the resolution of complex proteome samples by using two-dimensional offline LC/MS. The method applies a continuous salt gradient in the first separation dimension, which can significantly increase the number of identified proteins from complex samples due to higher chromatographic resolution compared to stepwise elution. In addition, the method offers high flexibility for the investigator since micro fractions of the first dimension can be stored, re-analyzed or chemically modified prior to further analysis. To achieve optimal resolution, the applied 2D LC method strongly requires optimization of the two separation dimensions. Especially elution gradients, analysis time, stationary phase material, and column dimension need to be designed and assembled adequately to attain a considerable increase in overall peak capacity. This Application Note shows data which influences the variables and components. It can serve as a guideline for the investigator to adapt his separation method for a specific proteome sample in order to achieve maximum peptide sequence information by MS/MS analysis. This will result in a maximum of identified proteins after a database search.
Brand: Agilent
File format: PDF
Size: 627 KB
MD5 Checksum: 257F266974FECA412E4A3D352F0064CA
Publication date: 02 July, 2012
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