Heather Callahan, Ph.D., Victoria Nieciecki, Eddie Adams, Ph.D.MO BIO Laboratories, a QIAGEN companyCarlsbad, CA, USA

IntroductionReversible immobilization of nucleic acids on silica-based supports such as spin columns, resins, and magnetic beads has streamlined the isolation and purification of DNA and RNA. Once considered a time consuming process, purified DNA and RNA is now readily acquired within minutes by individuals with minimal training. This method has become so universal that it is the basis of nearly every nucleic acid extraction kit commercially available. In stark contrast, streamlined sample preparation methods for proteomic studies of microbial populations has lagged behind. This technological delay has, in part, been due to the extraordinary structural and chemical diversity of protein populations and the distribution of those proteins at the subcellular level. These factors make determining the optimal extraction conditions for total microbial proteins difficult.

Important Considerations for

Silica Based Isolation of Total Microbial Proteins for Proteomic Applications

Challenges to total protein isolation are complex. Conditions must be established that not only encourage cell lysis and disruption of subcellular compartments, but also enable complete solubilization of proteins. Once solubilized, proteins must be kept stable in order to avoid their loss as a consequence of aggregation and unintended precipitation during subsequent handling steps. Proteins then need to be efficiently recovered in a way that eliminates time consuming and bias-inducing precipitation methods such as those consisting of trichloracetic acid (TCA), divalent metal ions, and alcohols.

In an effort to address these universal protein challenges, we developed a patent-pending method of total cellular protein isolation. We optimized a protocol that combines chemical and mechanical cell disruption with reversible immobilization of solubilized proteins to silica surfaces, such as silica spin columns. Once bound, proteins are washed and eluted into buffers that are suitable for 1D and 2D SDS-PAGE, proteolytic digestion and mass spectrometry. This reversible immobilization chemistry easily scales with throughput requirements, allowing the same chemistry to be applied to 96-well spin plates and silica-coated magnetic beads for use on liquid handling robotic instrumentation. Overall, the ease of use and scale-up ability of this method should greatly facilitate and accelerate protein isolation from complex microbial cells.

Materials and Methods The most important aspects of total protein extraction from cultured microbial cells involve simultaneous cell lysis and protein solubilization, followed by protein concentration and recovery. Mechanical lysis in the form of bead beating is the least biased means of cellular lysis and when performed in the correct lysis buffer, will instantaneously solubilize proteins. By picking the correct bead size and matching it with the best extraction buffer, cells walls of varying complexities can be easily lysed without the need for additional detergents or enzymes. We tested several lysis buffers for their ability to solubilize proteins from the gram negative bacterium, E. coli and the yeast, C. albicans. The best buffer was then paired with different bead types and sizes to optimize lysis for both microbes. All microbial cultures were grown overnight to stationary phase at 37°C. 1.8 mL of each culture was used per extraction. All results were visualized using Bio-Rad Any kD™ Mini-PROTEAN® TGX Stain-Free™ Gels and ran for 25 minutes at 200V.

Once the optimal lysis protocol was determined, total protein binding to silica-based spin filters was accomplished by a two-step binding process. In the first step, the crude lysate was combined with a neutral salt solution that immobilized salt-dependent proteins onto the silica matrix. In the second step, the flow through from the first step was combined with a buffered salt solution that then immobilized pH-dependent low molecular weight proteins onto the same column. Protein binding was also demonstrated on other silica matrices. After a water-based wash was used to remove impurities, immobilized proteins were eluted in a 1% sodium dodecylsulfate (SDS)/HEPES buffer. For SDS sensitive applications such as in-solution protease digestion and Filter-Aided Sample Preparation (1), buffers such as 7M urea/2M thiourea and the cleavable detergent, RapiGest™ (2%, Waters Corporation) were tested for their effectiveness at eluting proteins from the silica spin filter. The final developed protocol allowed complete sample processing in just 22 minutes (Figure 1).

To validate the protocol, eluted proteins from an E. coli culture were visualized by 1D SDS-PAGE alongside crude, unpurified lysate. Corresponding target windows at 35 - 50 kDa were excised and subjected to in-gel trypsin digestion. Digests were analyzed by nano LC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Data were searched using Mascot and parsed into Scaffold for validation and filtering to create a non-redundant list. The resulting protein identifications were classified into protein coding gene families using the on-line tool, PANTHER (2).

Figure 1. Flowchart of the protein extraction and purification protocol for cultured microbes

Results and DiscussionFor optimal protein solubilization a selection of chaotropic and kosmotropic buffers were evaluated. Of these, three buffers, a weak chaotrope, a strong chaotrope, and a detergent, had the best results. However, protein recovery was not consistent between the different cultured microorganisms (Figure 2). All three buffers were able to extract and solubilize E. coli proteins, while only the detergent and strong chaotrope were able to lyse and solubilize C. albicans proteins. Based on these results, we chose the strong chaotrope (C) as the basis for the microbial protein lysis buffer.

Figure 3. Lysis efficiency between various bead tube types for E. coli and C. albicans). The high chaotrope buffer was used as the lysis buffer for all samples.

To maximize lysis for both E. coli and C. albicans, we tested different sized beads and bead mixes with the strong chaotrope lysis buffer (Figure 3). The most effective bead type and size for E. coli was 0.1 mm glass with 0.5 mm glass being ideal for C. albicans (Lane F in Figure 3). Protein was obtained from all beads for both cultures, but the bead tube mix containing 0.1 mm and 0.5 mm glass was the best across both microbial cell types despite their differences in cell wall complexity.

Figure 5. E. coli proteins bound using the same two step binding system to either A) silica resin or B) silica spin filters. All samples were done in quadruplicate.

Figure 4. Binding efficiency of E. coli proteins with A) single neutral salt solution binding step and B) two step binding system consisting of the neutral salt solution, followed by a buffered salt solution. Note the missing low molecular weight proteins when only the neutral salt solution is added (A).

Due to the diverse nature of proteins, complete protein immobilization could only be accomplished through the use of two binding steps, with slight changes to the binding conditions. When only the neutral salt solution was added, low molecular weight proteins did not bind until a buffered salt solution with a more acidic pH was added in a second rebinding step (Figure 4).

We found this binding to be independent of the siliconized support structure itself, demonstrating good recovery of proteins from silica resin as well (Figure 5).

Figure 2. Comparison of the top three protein extraction buffers for E. coli and C. albicans. A = detergent, B = weak chaotrope, C = strong chaotrope

Figure 6. 1D SDS-PAGE gel highlighting the excised region for the silica spin column purified protein and the crude, unpurified protein that was trypsin digested and analyzed via nano LS-MS/MS.

Figure 7. Alternate silica spin filter elution buffers that could be used directly for in-solution and FASP protease digestion.

Table 1. Summary table of the proteins identified via mass spectrometry and their spectral counts for both the silica spin filter purified protein and the crude, unpurified protein.

Purified CrudeTotal number of proteins identified 388 397Total number of spectra matching 5433 5673Total number of unique peptides 3931 3883

Furthermore, when we evaluated other, more mass spectrometry-compatible elution buffers, we found urea/thiourea and the cleavable detergent, RapiGest™ to be comparable to 1% SDS/HEPES (Figure 6). 1% SDS/HEPES had the most optimal protein recovery but eluting with other buffers provides the benefit of eliminating the need for a buffer exchange column and allows the user to go directly to in-solution protease digestion or use the FASP method (1).

To demonstrate the efficiency of protein binding to silica spin filters, bound and eluted proteins were compared to crude, unpurified lysate by mass spectrometry. For both samples, the same 35 – 50 kDa area was excised, in gel trypsin digested and analyzed via nano LC-MS/MS (Figure 7).

The spin column purified sample demonstrated very efficient binding and recovery, showing only 2% difference in protein identifications compared to the crude, unpurified lysate (Table 1).

Similarly there was good correspondence between the PANTHER generated protein classifications with the differences being primarily in the number of proteins represented in several of the families (Figure 8).

Figure 8. Panther generated protein class relationships based on E. coli proteins excised and digested within the 35 – 50 kDa range.

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NoviPure® Microbial Protein Kit Crude, Unpurified Proteins

** Chart tooltips are read as: Category name (Accession): # genes: Percent of gene hit against total # genes; Percent of gene hit against total # Protein Class hits

ConclusionsMicrobial proteins can be quickly and easily purified on silica based supports, similar to commercially available DNA and RNA isolation kits that are used so widely in life science research. We have demonstrated that by combining a denaturing chaotrope with a mixed bead tube, we can optimize the lysis of cultured microorganisms that represent a range of cell wall complexities. The optimized lysis buffer also serves to limit bias by maximizing the solubility of proteins that differ in their hydrophobicity/hydrophilicity, isoelectric points, and pH sensitivity. The most novel aspect of our method is a two-step immobilization chemistry that makes it possible to bind, wash, and elute a diverse population of proteins without sacrificing yield and without introducing additional bias. Eluted proteins are then ready to use in a variety of proteomics applications.

References1. Universal sample preparation method for proteome analysis. Jacek R Wis´niewski, Alexandre Zougman, Nagarjuna Nagaraj and Matthias Mann. Nature Meth (2009), Vol 6 (5): p 359-62.

2. PANTHER version 10: expanded protein families and functions, and analysis tools. Huaiyu Mi, Sagar Poudel, Anushya Muruganujan John T. Casagrande and Paul D. Thomas. Nucl. Acids Res. (2016) doi: 10.1093/nar/gkv1194.