In preparation for mass spectrometry analysis, additives such as chaotropic agents often are used. These compounds denature and solubilize proteins, rendering more of their structure accessible to trypsin, the most commonly used proteolytic enzyme. Urea and guanidine hydrochloride are the traditional chaotropic agents used. At the high concentration required for efficient denaturation, however, urea and guanidine also inhibit trypsin activity(1).
The concentration of chaotropic reagents is, therefore, commonly reduced before the addition of trypsin. However, this routine procedure is inadequate, because dilution of the aforementioned reagents permits the proteins to refold and hydrophobic proteins to aggregate.
The main obstacle for mass spectrometry analysis is maintaining the protein in a denatured state throughout the digestion and separation process. While this can be achieved through common detergents, such as sodium dodecyl sulfate (SDS) and Triton®-X, these detergents are not suitable for mass spectrometry analysis, because they can decrease protease activity, suppress peptide ionization and interfere with chromatographic separation(0)(3). To avoid these drawbacks, sample preparations with detergents require additional detergent removal manipulations, which potentially can lead to significant sample loss.
The main obstacle for mass spectrometry analysis is maintaining the protein in a denatured state throughout the digestion and separation process.
To alleviate the risk of sample loss, many mass spectrometry-compatible surfactants have been developed and are now commercially available. In this article, we compare two of these surfactants, Invitrosol™ (Invitrogen) and ProteaseMAX™ Surfactant, Trypsin Enhancer (Promega) for their ability to increase proteomic coverage of rat brain homogenate with MudPIT (Multidimensional Protein Identification Technology).
One hundred micrograms of rat brain homogenate in 0.32M sucrose and 4mM HEPES (pH 7.5) was centrifuged at 17,000 x g at 4°C for 15 minutes. After the supernatant was removed, the pellet was solubilized either in 100µl of Invitrosol™ with 8M urea or in 50µl of 0.2% ProteaseMAX™ Surfactant, Trypsin Enhancer (Cat.# V2071) and 50µl of 8M urea. Four membrane pellets were solubilized with each reagent. Tris(2-Carboxyethyl) phosphine hydrochloride (TCEP) was added to solution at a final concentration of 5mM to reduce the disulfide bonds. The samples then were incubated at 60°C for 10 minutes followed by vortexing for one hour. The samples were incubated at 60°C for 10 minutes, then sonicated for one hour. Iodoacetamide was added to the samples at a final concentration of 10mM for protein alkylation, and the samples were incubated at room temperature in the dark for 20 minutes. The volume of the samples was increased to 250µl with 100mM Tris (pH 8.0), 2mM calcium chloride for the Invitrosol™ reagent and with 50mM ammonium bicarbonate for the ProteaseMAX™ Surfactant samples.
Four micrograms of trypsin was added to each digestion. In addition, 2.5µl of 1% ProteaseMAX™ Surfactant was added to the ProteaseMAX™ Surfactant samples. All samples were then placed in a shaking incubator at 37°C. The Invitrosol™ samples were incubated overnight, while the ProteaseMAX™ Surfactant samples were incubated for three hours. Following the digestion, the samples were placed in a –80°C freezer until mass spectrometry analysis.
On the day of analysis, samples were thawed at room temperature, and formic acid was added to a final concentration of 5%. The samples were centrifuged at 17,000 x g at room temperature for 30 minutes. The supernatant was placed in a new tube. MudPIT analysis was performed as previously described(4). Briefly, the samples were pressure loaded on a 250µm i.d. capillary with a Kasil frit containing 2.5cm of strong cation exchange (SCX) 5µm resin and 2.5cm of 10µm reverse phase (RP) resin. After sample loading, the column was desalted with buffer containing 95% water, 5% acetonitrile and 0.1% formic acid. After desalting, a 100µm i.d. capillary with a 5µm pulled tip packed with 15cm of 4µm RP resin was attached to the desalting column with a union. The entire split-column (desalting column–union–analytical column) was placed inline with an Agilent 1100 quaternary HPLC and analyzed using a modified 12-step separation described previously(4). As peptides eluted from the capillary column, they were electrosprayed directly into an LTQ 2-dimensional ion trap mass spectrometer (ThermoFisher, San Jose, CA) with the application of a distal 2.4 kV spray voltage. A cycle of one full-scan mass spectrum (400–1,400 m/z) followed by 6 data-dependent tandem mass spectra was repeated continuously throughout each step of the multidimensional separations.
All tandem mass spectra were collected using normalized collision energy (a setting of 35%), an isolation window of 3 m/z, and 1 µscan.
Four MudPIT runs were analyzed for each surfactant. The data was searched against a rat IPI database using SEQUEST(5). The reverse rat database was also searched to estimate the false discovery rate(6). The false discovery rate was less than 1% at the protein level for each MudPIT run. In addition, we required at least two peptides for all protein identifications. Overall, we observed a greater number of proteins and peptides per MudPIT analysis with ProteaseMAX™ Surfactant compared to Invitrosol™. The average number of identified proteins was 1,699 and 2,251, and the average number of peptides was 9,621 and 15,423 for the Invitrosol™ and ProteaseMAX™ Surfactant samples, respectively. Thus, the ProteaseMAX™ Surfactant samples had a 32% and 60% increase in protein and peptides, respectively. In total, we identified 3,315 nonredundant proteins from all the MudPIT analyses. There were 2,286 proteins identified in both the Invitrosol™ and ProteaseMAX™ Surfactant samples. Four hundred seventy-two proteins were identified only in the Invitrosol™ samples and 1,053 proteins identified only in the ProteaseMAX™ Surfactant samples. Among the proteins identified in both samples, 1,680 proteins had a greater sequence coverage in the ProteaseMAX™ Surfactant samples, while 460 proteins had a greater sequence coverage in the Invitrosol™ samples.
Figure 1. Protein identifications from MudPIT analyses.
Brain homogenate prepared with either Invitrosol™ or ProteaseMAX™ Surfactant was analyzed four times by MudPIT for a total of eight MudPIT experiments. Panel A. The total number of proteins identified by at least two peptides in each MudPIT experiment. Panel B. The total number of peptides identified in each MudPIT experiment.
Next, we compared the number of integral membrane proteins identified with each sample preparation. Using the TMHMM algorithm(7), 535 (20%) and 694 (21 %) proteins identified using Invitrosol™ and ProteaseMAX™ Surfactant, respectively, were predicted to have at least one transmembrane domain. ProteaseMAX™ Surfactant increased the number of identified membrane proteins with one and multiple transmembrane domains. For example, ProteaseMAX™ Surfactant identified 28.9% more membrane proteins with one transmembrane domain, and 87.5% more membrane proteins with 7 transmembrane domains compared to Invitrosol™. There were 381 transmembrane proteins identified with both reagents.
Figure 2. Comparison of the number of integral membrane proteins identified with each sample preparation.
The number of identified proteins with a predicted membrane-spanning region was determined by TMHMM.
ProteaseMAX™ Surfactant resulted in greater sequence coverage of 342 of these proteins. For example, the glutamate receptor 2 (GluR2) is membrane protein with transmembrane regions that binds glutamate at the synapse. With Invitrosol™, GluR2 was identified with 13 peptides, representing 20.4% sequence coverage, while with ProteaseMAX™ Surfactant treatment GluR2 was identified with 34 peptides, representing 42.8% sequence coverage. One hundred fifty-four and 313 membrane proteins were identified solely in the Invitrosol™ or ProteaseMAX™ Surfactant samples,respectively.
Our observed increase in protein identifications with ProteaseMAX™ Surfactant may be attributed to two factors. First, ProteaseMAX™ Surfactant may be more efficient at solubilizing a complex mixture than Invitrosol™, resulting in more proteins that are accessible to trypsin. Secondly, during any sample preparation, there is always some degree of sample loss with each step. It is, therefore, possible that ProteaseMAX™ Surfactant aids in protein or peptide recovery. Although the data show advantages of ProteaseMAX™ Surfactant versus Invitrosol™ reagent, we argue that both surfactants could be employed in parallel to achieve the highest proteome coverage. Our suggestion is based on the observation that either surfactant allows for identification of unique proteins not found with other surfactant.