Automated protein preparation techniques using a digest robot.
Since the introduction of fast analysis methods for peptide mixtures such as MALDI-MS, peptide micropreparation and digest methods have become an important bottleneck in the protein characterization process. We therefore developed and describe here a digest robot capable of processing 30 protein samples in parallel [Houthaeve et al. (1995), FEBS Lett. 376, 91-94]. Briefly, after gel pieces or blots are cut out, they are loaded in flowthrough reactors and these are loaded in a thermocontrolled reactor block. The proteins are then washed, reduced, and alkylated, proteolytically or chemically cleaved, and resulting peptides extracted. The system allows the parallel use of different reagents and enzymes during the same run, and is compatible with RP-HPLC peptide separation and Edman degradation, MALDI-MS, and NanoES-MS/MS. The digest robot is now also commercially available from ABIMED. In an ongoing project aimed at elucidating proteinaceous structures involved in the functional and structural maintenance of the Golgi apparatus, we illustrate the strength of the digest robot for the fast analysis of several proteins. We conclude that the performance of the digest robot is comparable to currently used manual digestion methods. The approach outlined makes sample preparation procedures faster, simpler, and less labor-intensive.
Houthaeve
T
T
Protein and Peptide Group, EMBL, Heidelberg, Germany. Tony.Houthaeve@RUG.AC.BE
Gausepohl
H
H
Ashman
K
K
Nillson
T
T
Mann
M
M
D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006056:Golgi Apparatus / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D011506:Proteins / Q000032:analysis* / Q000502:physiology; D012371:Robotics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D011506:Proteins