Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and other alternative methods of mass spectrometry (MS) analysis have become essential methods of protein analysis(1)(2). Using MS methods researchers identify proteins, peptides(3)(4), post-translational modifications(5)(6)(7), protein profiles, protein:protein interactions, protein:small molecule interactions(8)(9) and study protein structure and function(10). Polyhistidine-tagged protein purification systems provide large amounts of a protein or small amounts of multiple proteins for study. However, the elution buffers used in these systems contain salts (e.g., imidazole) that cannot be used in mass spectrometry analysis. To be compatible with MALDI–TOF MS analysis, eluted samples need to undergo tedious dialysis methods or size exclusion separation techniques to remove salts. Previously we have described a method of eluting polyhistidine-tagged proteins using the MagneHis™ Protein Purification System (Cat.# V8550) for analysis using mass spectrometry(0). In this article, we describe a method for the elution of a polyhistidine-tagged protein from HisLink™ Protein Purification Resin using 40% ethanol or 0.1% TFA in 50% acetonitrile. These elution conditions allow direct MS analysis and provide clean mass spectrometry data necessary for high-throughput analysis using MALDI-TOF MS.
Preparation of Polyhistidine-Tagged Proteins for MALDI-TOF Mass Spectrometry Analysis
Polyhistidine-tagged calmodulin was purified from E. coli strain BL-21 (DE3) using HisLink™ Resin as described in the HisLink™ 96 Protein Purification System Technical Bulletin (#TB342). Wash and elution conditions were optimized to decrease substances that interfere with mass spectrometry analysis. The resin/protein complex was first washed twice with 500µl of 100mM HEPES (pH 7.5) plus 0.5M NaCl to decrease nonspecific binding to the resin. The particles were then washed four times with Nuclease Free Water (Cat.# P1193) to remove the NaCl and buffer from the resin. The protein was eluted from the resin using either 0.1% TFA in 50% acetonitrile or 40% ethanol. Samples were dried using a SpeedVac® DNA 110 Concentrator and were resuspended in 200µl of Nuclease-Free Water. A portion of the rehydrated samples was removed for protein gel analysis, and the remainder was dried and sent to HT Laboratories (San Diego, CA) for MALDI-TOF MS analysis.
Results and Conclusions
Gel analysis showed that the protein of interest (His-calmoulin) was eluted from the resin using the various elution conditions (Figure 1). The amounts of protein recovered using the alternative methods appear to be less than the MagneHis™ Elution Buffer (control); however, the protein amount is sufficient for MALDI-TOF MS analysis. MALDI-TOF MS data show the MagneHis™ Elution Buffer (containing imidazole) causes extensive background interference (Figure 2); however the samples eluted with 40% ethanol or 0.1% TFA in 50% acetonitrile show clean protein peaks well above background (Figures 3 and 4).
Polyhistidine-tagged proteins eluted using conventional elution buffers contain substances that interfere with mass spectrometry analysis. By modifying the wash and elution conditions necessary to purify polyhistidine-tagged proteins, analysis using mass spectrometry is achieved easily and efficiently. Results generated from the alternative elution conditions have the defined protein peaks and reduced background that are necessary qualities for successful MALDI-TOF MS protein analysis.
Figure 1. Comparison of the amount of polyhistidine-tagged protein recovered using different elution conditions.
For each rehydrated sample, 50µl of sample was combined with 20µl SDS loading buffer. This mixture was heated at 95°C for 5 minutes. The prepared samples (20µl) were loaded and run on a 4–20% Tris-glycine gel (Invitrogen). The gel was stained in SimplyBlue™ Safe Stain (Invitrogen). Lane M, Protein marker; Lane 1, MagneHis™ Elution Buffer (0.5M imidazole); Lane 2, 40% ethanol; Lane 3, 0.1% TFA in 50% acetonitrile.
Figure 2. MALDI-TOF analysis of polyhistidine-tagged calmodulin eluted with MagneHis™ Elution Buffer showing background interference.
Figure 3. MALDI-TOF analysis of polyhistidine-tagged calmodulin eluted with 0.1% TFA in 50% acetonitrile.
Protein eluted with 0.1% TFA in 50% acetonitrile showed decreased background interference compared to protein eluted with MagneHis™ Elution Buffer (Figure 2).
Figure 4. MALDI-TOF analysis of polyhistidine-tagged calmodulin eluted with 40% ethanol.