Sample preparation and in-solution protease digestion of proteins for chromatography-based proteomic analysis.
The adoption of chromatography-based proteomics approaches has resulted in the development of new methods for optimal use of these technologies. One such technology, named multidimensional protein identification technology (MudPIT), directly couples liquid chromatography with tandem mass spectrometry. In the MudPIT approach, digested protein samples are directly loaded onto a microcapillary column packed sequentially with reversed-phase and strong cation exchange resins. Once digested protein samples are loaded onto the column, the column is placed in-line between a high-performance liquid chromatography system and an electrospray ionization tandem mass spectrometer (ESI-MS/MS). The digested protein samples for MudPIT analysis must be directly compatible with ESI-MS/MS, so the sample preparation and digestion protocols must be optimized for this purpose. The primary objective of all of the protocols in this unit is to yield a final digested protein mixture of less than 300 microl that can then be directly loaded onto a MudPIT column.
Washburn
Michael P
MP
Stowers Institute for Medical Research, Kansas City, Missouri.
D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000737:chemistry*; D006801:Humans; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
D011506:Proteins; D020543:Proteome; D010450:Endopeptidases