In recent years, mass spectrometry (MS) has become an indispensable proteomics tool(1)(2). Because of their ease of purification and lack of interference with protein function, His-tagged proteins have been combined with mass spectrometry in studies of structure and function(3), protein-protein interactions(4), post-translational modifications(5)(6)(7), molecular weight determination(8) and peptide mapping. Unfortunately, methods normally used for His-tagged protein purification result in the presence of imidazole, and high concentrations of salt and detergents, which interfere with mass spectrometric analysis by causing high backgrounds (Figure 1, Panel A). However, modified washing and elution conditions can decrease the high mass spectrometry backgrounds for His-tagged proteins, as shown in Figure 1, Panels B (– DNA) and C (+ DNA). In this article we describe a method developed for the elution of His-tagged proteins from MagneHis™ Particles that reduces the salt and eliminates imidazole, so that His-tagged proteins can be analyzed more precisely by mass spectrometry.
Figure 1. MALDI-TOF analysis of MagneHis™ purified His-tagged GFP.
A T7 S30-based bacterial cell-free system was used in this experiment. In vitro translated His-tagged GFP was purified using the procedure described in the article. Panel A: His-tagged GFP purified with 0.5M imidazole-containing buffer. Panel B: Control reaction without His-tagged GFP DNA. Panel C: His-tagged GFP DNA purified using the modified wash and elution steps.
MALDI-TOF Analysis of MagneHis™ Purified Proteins
A protocol for preparation of His-tagged proteins for MALDI-TOF analysis, is shown in Table 1. This method can be used for the purification and elution of His-tagged proteins from bacterial cells or cell-free bacterial expression systems. Steps 1–3 describe the purification of His-tagged proteins as directed in the MagneHis™ Protein Purification System Technical Manual, #TM060, while Steps 4–7 describe a modified wash and elution protocol that reduces salt and eliminates imidazole.
Table 1. Preparation of MagneHis™ Purified Proteins for MALDI-TOF Analysis.
A comparison of the standard protocol (TM060 and Table 1, Steps 1–3) and the modified protocol (Table 1, Steps 4–7) for purification/elution of His-tagged Green Fluorescent Protein (His-GFP) expressed in a T7 S30 cell-free expression system and analyzed by MALDI-TOF, is shown in Figure 1, Panels A and C. Panel A is the MALDI-TOF spectrum of the His-GFP purified/eluted with the standard protocol (containing imidazole and high salt concentrations), whereas Panel C shows the MALDI-TOF spectrum of the His-GFP purified/eluted with the modified protocol (Table 1, Steps 4–7). Background peaks are reduced when using the modified protocol in Steps 4–7 of Table 1. SDS-PAGE analysis is shown in Figure 2 (– DNA in Lane 1, + DNA in Lane 2) using the modified protocol for purification/elution. The data in Figure 2 show that using the modified protocol, MALDI-TOF analysis is possible for His-tagged proteins expressed in a bacterial cell-free expression system.
Figure 2. SDS-PAGE analysis of His-tagged GFP purified from a T7 S30 bacterial cell-free system using the modified protocol.
Lane 1: In vitro coupled transcription and translation of a sample without DNA. Lane 2: In vitro coupled transcription and translation with His-tagged GFP DNA.
The development of a sample preparation method for His-tagged proteins purified using MagneHis™ Protein Purification System can drastically reduce the background levels in MALDI-TOF mass spectrometry analysis. This method can be used for the analysis of His-tagged protein expressed in bacteria in vivo as well as in bacterial cell-free expression systems.