Identification of Replication Protein by Nanoelectrospray Time-of-Flight Mass Spectrometry
Introduction
Nanoelectrospray (nanoESI) has become a powerful tool in bioanalytics and is now used as a routine analytical method1. The advantages of nanoelectrospray as compared to conventional electrospray (ESI) include very low flow rate and more tolerance toward salt contamination in the analyte solution2. Thus, a few μL of analyte solution suffice for extended mass spectrometric studies.
This applications report demonstrates the use of nanoESI for protein identification. A commercially available replication protein A3 is in-gel digested with trypsin and desalted with ZipTip C18 tip. The analysis is performed using nanoESI coupled with the AccuTOF™ time-of-flight MS system to obtain the peptide fingerprint followed by a database search with ProFound3 software.
Experimental
The protein sample A3 was trypsin in-gel digested and extracted with the buffer solution containing 40 mM ammonium bicarbonate, 50% acetonitrile and 0.1 % TFA, and then dried using SpeedVac. The dried peptides were reconstituted with 0.1 % formic acid solution and desalted with ZipTip C18 tip and eluted with 10 µL acetonitrile/0.1% formic acid solution (50/50). 2 µL of the eluent was applied to the spray needle (New Objective).