Transfection of cells is one of the main techniques used to influence gene expression. Most primary cells and human skeletal myoblasts (SkMC) in particular are very difficult to transfect, whereas for cell lines such as C2C12, many suitable transfection reagents and protocols are available. ... more
Enzymatic Protein Digest for Mass-spectrometric AnalysisKairat Madin*, Sylvia Hermann, Claudia Kirr, Doris Schmitt, and Thomas Koch
Roche Applied Science, Penzberg, Germany
The main focus in proteomic studies is on the identification of proteins in given biological samples. Proteins isolated and separated from a given sample (e.g., whole cell lysates, blood or tissue, protein complexes) by immunoprecipitation or affinity chromatography or two-dimensional electrophoresis must be proteolytically cleaved in the course of sample preparation for identification and characterization. A reproducible cleavage pattern of digested proteins is a prerequisite for the unambiguous identification of these proteins by mass spectrometry (MS). The objective of the study described in this article was to demonstrate the applicability of sequencing-grade endoproteases to mass-spectrometric analysis of proteins. Here we show results of MS analysis of two proteins employing the most frequently used sequencing-grade endoproteases such as trypsin, Lys-C, Asp-N, and Arg-C. It was observed that the sequencing-grade proteases used in this study are excellent tools for MS analysis of proteins based on high specificity, purity, and lot-to-lot consistency.
Materials and Methods
Lysozyme, recombinant green fluorescent protein (rGFP), endoproteases Lys-C, Arg-C, Asp-N (all sequencing grade), and trypsin (proteomics grade), and other critical reagents were from Roche Applied Science.
Lysozyme and rGFP were dissolved in water to a final concentration of 1 mg/ml. For reductive alkylation, protein samples were reduced with dithiotreitol and subsequently alkylated with iodoacetamide. For digestions, routinely 1 µg of endoprotease was mixed with 100 µg of protein sample (1:100 ratio). To test for contaminating endoproteases, a tenfold excess of endoprotease was used (1:10 ratio). Digestion reactions were incubated routinely for 16 hours at 37°C, and for only 1 hour in experiments with shortened incubation time. For stability tests, endoproteases were incubated without substrate, for 24 hours at 25°C. After addition of substrate, incubation was continued for another 16 hours at 37°C.
Lysozyme and rGFP samples were denatured and solubilized for isoelectrofocusing by diluting protein samples with 8 M urea, 2% CHAPS, reducing agent, traces of bromophenol blue, and appropriate ZOOM carrier ampholites (Invitrogen). 140 µl of rehydration solutions containing protein sample were passively incubated with ZOOM gel stripes for 1 hour. IEF was performed in the ZOOM IPGRunner system with ZOOM Dual Power power supply.
Then IEF strips were applied to a second-dimension 4%–12% Bis-Tris ZOOM gel, and run under standard conditions. Spots from second-dimension gels were identified by staining with SimplyBlue™ SafeStain, excised and discolored with 0.1 M ammoniumhydrogencarbonate in 30% acetonitril followed by reductive alkylation. The in-gel proteins were digested in the dark with 0.28 µg of endoproteases per sample at 37°C for 16 hours. Peptides were extracted with a solution containing 20% acetonitril, 0.1% TFA and 1.3 mM Tris, and subjected to MALDI analysis.
MALDI-TOF mass spectrometry
Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectra were acquired on a Bruker Reflex III instrument (Bruker Daltonics) in the reflector positive ion mode. Prior to MALDI ana-lysis the samples were desalted using ZipTip™µ-C18 pipette tips (Millipore) and eluted directly onto the MALDI target using MALDI matrix (alpha-cyano-4-hydroxycinnamic acid, 10 mg/ml in 70% acetonitril, 0.03% TFA). Identification of mass profiles was achieved by comparing the peptide masses obtained with theoretically expected data generated using PeptideMass software (http://us.expasy.org/tools/peptide-mass).
Results and Discussion
The main focus in proteomic analyses is on the identification of proteins. Digestion of protein samples in solution or in gel coupled with modern mass-spectrometric analysis is a powerful tool for analysis of complex protein mixtures. In general, mass spectrometry experiments consist of five major common steps: 1. isolation of protein; 2. enzymatic digest of proteins to peptides; 3. sample preparation for MS analysis; 4. generation of the MS spectrum, and 5. identification of obtained peptide mass profile by comparison against protein sequence database or predicted reference peptide masses. It is extremely important that proteases used for peptide mapping and for characterization of protein structural domains have to be of well-defined cleavage specificity and free of contaminating activities such as unspecific proteases. Therefore, proteome research demands the use of specific endoproteases of highest purity.
Sequencing-grade endoproteases from Roche Applied Science are highly purified enzymes since they are subjected to extensive purification to remove contaminating protease activities. They retain consistently high activity during long digestion procedures. Endoproteases Asp-N, Lys-C, Arg-C, and Trypsin are most frequently used for enzymatic protein digestion.
The goal of this set of experiments was to show applicability of sequencing-grade endoproteases to mass-spectrometric analysis of proteins. We intended to test the suitability of endoproteases for in-solution and in-gel digest and to confirm high purity and stability of enzymes.
To demonstrate the stability of endoproteteases we pre-incubated enzymes in solution for 24 hours at 25°C before lysozyme or rGFP were added in 1:100 ratio. After addition of the substrate, the digest reactions were incubated for another 16 hours at 37°C. Following MALDI-TOF analysis of lysozyme digested with endoprotease Asp-N the identified peaks and the predicted matched sequences were compared. As shown in Figure 1, 100% sequence coverage was obtained. In the case of lysozyme we achieved 100% sequence coverage also with Lys-C and Arg-C, and 95% with trypsin. In the case of rGFP as a substrate we reached the following sequence coverage values: 86%, 72%, and 79% for Lys-C, Asp-N, and trypsin, respectively.
We also performed digests of rGFP under standard conditions with trypsin, Lys-C, and Asp-N in 1:100 ratio. As can be seen in Figure 2 for rGFP, we achieved 77% sequence coverage using trypsin, and 72% and 78% using Asp-N and Lys-C, respectively. In order to test for contamination in endoproteases we incubated lysozyme and rGFP with a tenfold excess of endoproteases (1:10 ratio). MS analysis revealed no additional peaks (data not shown).
To further demonstrate effectiveness of the endoproteases we performed additional experiments with shortened incubation time using trypsin and Lys-C. Digests were prepared and analyzed after only 1 hour of incubation. As a result, 100% sequence coverage was achieved in both experiments and with both endoproteases (Figure 3).
To finally demonstrate the suitability of endoproteases for in-gel digest, we subjected lysozyme and rGFP to 2-D electrophoresis. Gel spots were excised, destained and prepared for the digest reaction. Digest reactions were incubated on a shaker for 16 hours (overnight) in the dark. The extraction solution was then added and after centrifugation peptide digests were analyzed by MALDI. Figure 4 illustrates the results for lysozyme digested with trypsin and shows that the expected peptide mass spectra matched with the peaks predicted by PeptideMass software. Sequence coverage values with other enzymes were: 84% for trypsin and Lys-C, 100% for Asp-N, and 98% for Arg-C. For rGFP the sequence coverage was 75% for trypsin and 74% for Lys-C.
In the current study, it was clearly demonstrated that the endoproteases trypsin, Lys-C, Asp-N, and Arg-C from Roche Applied Science have high specificity and qualify for mass-spectrometric analysis. They are free of impurities and serve as valuable analytical tools generating peptides for identification of proteins under controlled conditions and with predictable results. They ensure high stability and reduce sample preparation time since preparation of reconstitution buffer is not required.
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