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We have recently improved the automation of an in-gel digestion system, DigestPro 96, using in situ alkylation of proteins with acrylamide, conducted during one-dimensional (1D) SDS-PAGE. The improved method included the processes of destaining, dehydration, trypsin digestion, and extraction but excluded the reduction and alkylation steps following staining of proteins with CBB. The extracted peptide mixtures were directly loaded onto a micro C18 LC column of the mass spectrometer. The resultant spectra were processed with “Mascot” search engine to estimate the sequence coverage of the bovine serum albumin (BSA). The original method, designed for Laemmli 1D SDS gel applications, consisted of reduction and post-alkylation with iodoacetamide, which produced carboxyamidemethyl (CAM; –S–CH2CONH2) derivatives. The original method also included a desalting step essential for mass spectrometry, especially matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We compared the original and improved methods using BSA (3 pmol loaded to the gel, one third of digested peptide mixture injected into LC-MS). The original method yielded both CAM and propionicamide (PAM; –S–CH2CH2CONH2) derivatives. The source of PAM derivatives is the unpolymerized acrylamide formed during electrophoresis. The sequence coverage of CAM derivatives of BSA by the original method was 10% with desalting and 19% without desalting. The sequence coverage of PAM derivative by the improved method was 32%. Our results clearly show the advantage of our improved automated in-gel digestion method for in situ PAM alkylated protein with respect to peptide recovery, compared with the original method with CAM post-alkylation.
In-gel digestion of protein is a widely used procedure in proteomics studies. However, the procedure is tedious and susceptible to contamination. Automation of the procedure1,2 has simplified the assay and reduced potential contamination. The automated protocol comprises several steps including destaining, reduction, alkylation (using –S–CH2CONH2 [carboxyamidemethyl; CAM]), digestion and extraction of the tryptic peptides. Among the steps of the automated protocol, reduction and alkylation are performed so that the cysteinyl peptide yields CAM derivatives. The cysteinyl residues, however, could also react with unpolymerized acrylamide monomer present in the electrophoresis gel. The reaction occurs during the electrophoresis run and produces propionic amide (PAM) derivatives, which are stable byproducts. The formation of PAM sometimes hinders fragment search procedure that expects CAM derivatives to be present. Mineki et al.3 have considered this by-product (PAM derivative) to be the major and sole “S”-containing peptide for mass spectrometry (MS) analysis. The digestion method described by Mineki et al.3 does not include reduction, alkylation, and desalting procedures. In this article we report improvement of the automated in-gel digestion protocol.
Molecular marker proteins of a wide molecular mass range (6.5–200 kDa) were obtained from Bio-Rad (Hercules, CA). The marker was run with one-dimensional sodium dodecyl sulfate (1D SDS) electrophoresis so that a band of 3 pmol of bovine serum albumin (BSA) was present in one lane.
Except for testing the protocol recommended by the supplier of the instrument, the protein was in situ alkylated during the electrophoresis run using the method described by Mineki et al.3 Briefly, sample were added to sample buffer containing 10% glycerol, 2% SDS, 0.1 M dithioerythritol (DTE) and 0.0025% bromphenol blue. The sample was then incubated at 95°C for 3 min. After cooling the sample to room temperature, a 4.7-μL aliquot of 30% acrylamide solution (20 μmol) was added. Then, the sample solution was loaded onto a gel and run.
A 12-lane, 130 × 130 × 1-mm SDS gel was prepared with 10% separation gel and 4% stacking gel acrylamide containing 0.87% and 0.35% piperazine diacrylamide, respectively. The gel was allowed to stand overnight at 4°C, and then pre-run at 20 mA for 1 h before loading the sample. After loading, proteins were concentrated in the stacking gel at 5 mA for 50 min and were resolved in the separation gel at 20 mA for 2 h. Staining was performed using CBB R-350 (Pharmacia & Upjohn Diagnostics, Uppsala, Sweden) or Plus One silver nitrate reagent free of glutardialdehyde (Pharmacia & Upjohn) according to the method of Mineki et al.3
The BSA band was cut and processed by an automatic in-gel digestion instrument (DigestPro96, Intavis AG, Koeln, Germany). The original method supplied by the manufacturer is shown in Figure 11.. A gel run by Laemmli’s method4 was used for this protocol. The improved method for CBB-stained gel is shown in Figure 22,, and the modified method of silver-stained gel in Figure 33.. The manual digestion method employed was the one described by Mineki et al.3
Mass spectrometry analysis was performed with AB-QSTAR pulsar i hybrid (Applied Biosystems, Framingham, CA) combined with a microliquid chromatograph (Magic 2002; Michrom Bioresources, Abum, CA) equipped with 0.2-mm ID × 50 mm Magic C18 column. In order to evaluate the improved method of DigestPro96, the sequence coverage of BSA by MS/MS was obtained with automatic search using “Mascot” software (Matrix Science, London, UK). The sequence coverage was calculated in the “Mascot” software as (total number of the amino acid residues of identified sequences) / (total number of amino acid residues of BSA).
The BSA sequence coverage analysis by Mascot software was performed using CAM derivatives and also using passively coexisting PAM derivatives. The coverage was 10% for CAM derivatives and 16% for PAM derivatives. PAM derivatives were formed during electrophoresis and are shown in Table 11.. The sequence coverage of the method, which did not include desalting with Zip Tip, was 19% with CAM derivatives and 27% with PAM derivatives. These results indicate that the recovery of peptides from the desalting column was 50–59%. Small-sized hydrophilic peptides seemed to pass through the Zip Tip while the large hydrophobic peptides were retained on the column (Figures. 4 and 55).). This resulted in a marked decrease in the recovery of peptides.
With regard to the improved method using in situ alkylation with acrylamide, the sequence of BSA identified by Mascot software was limited to PAM derivative. The method provided 32% coverage of BSA. Compared with the original method, three processes were omitted in the improved method: reduction, alkylation of stained proteins before in-gel trypsin digestion, and desalting before mass spectrometric analysis.
In the next step, we compared the BSA sequence coverage obtained by the manual protocol with that obtained by the improved (automated) method. Both protocols employed in situ alkylation with acrylamide during electrophoresis. The sequence coverage of the manual protocol was 48% and that of the automated instrument was 32%. The latter represents 66% of the sequence coverage of the manual protocol. Taking into consideration the relatively small amount of protein (3 pmol) that was digested, our results indicate a significant improvement by the automated protocol.
Next we compared the sequence coverage of BSA stained with CBB with the coverage obtained with silver nitrate reagent (Table 22).). The protocol used in both studies was the automated protocol employing in situ alkylated proteins.
The sequence coverage of BSA stained with silver nitrate was 21%, which represented 66% of the coverage of BSA stained with CBB. Although the sequence coverage on silver staining was lower, the improved protocol with silver staining was still considered to be usable.
We also analyzed the detection limit of the modified automated protocol for silver staining (Table 22).). The detection limit was 750 fmol with a BSA sequence coverage of 11%.
In this report, we describe the use of a modified automated in-gel digestion protocol for proteins using in situ alkylation with acrylamide. The sequence coverage of BSA using the modified protocol was 32%, which was threefold higher than that of the original method (10%). The improvement is probably due to PAM derivatization during electrophoresis, elimination of reduction and alkylation processes before in-gel digestion with trypsin, and deletion of the desalting process before mass spectrometric analysis. The sequence coverage of BSA by the improved protocol exhibited 66% value of the corresponding manual protocol in the digestion of the 3-pmol sample. Thus, the automated protocol seems to be useful for routine protein digestion. The protocol of the silver-stained gel showed 21% sequence coverage, which corresponded to 66% of the value of CBB-stained gel. The detection limit of the protocol was 750 fmol.