Comparison of In-Solution, FASP, and S-Trap Based Digestion

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Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies Katelyn R Ludwig, Monica M Schroll, and Amanda B Hummon J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00235 • Publication Date (Web): 13 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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Journal of Proteome Research

Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies

Katelyn R. Ludwig,1,2 Monica M. Schroll,1,2 and Amanda B. Hummon2,*

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Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA

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Department of Chemistry and Biochemistry and the Comprehensive Cancer Center, The Ohio

State University, Columbus, OH, USA. * Corresponding author. Amanda B. Hummon: Tel.: +1 614-688-2580. E-mail address: [email protected]

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Abstract Bottom-up proteomic strategies rely on efficient digestion of proteins into peptides for mass spectrometry analysis. In-solution and filter-based strategies are commonly used for proteomic analysis. In recent years, filter-aided sample preparation (FASP) has become the dominant filter-based method due to its ability to remove SDS prior to mass spectrometry analysis. However, the time-consuming nature of FASP protocols have led to the development of new filter-based strategies. Suspension traps (S-Traps) were recently reported as an alternative to FASP and in-solution strategies as they allow for high concentrations of SDS in a fraction of the time of a typical FASP protocol. In this study, we compare the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. We performed label-free quantification to analyze the differences in the portions of the proteome identified using each method. Overall, our results show that each digestion method had a high degree of reproducibility within the method type. However, S-Traps outperformed FASP and in-solution digestions by providing the most efficient digestion with the greatest number of unique protein identifications. This is the first work to provide a direct quantitative comparison of two filter-based digestion methods and a traditional insolution approach to provide information regarding the most efficient proteomic preparation. Keywords: sample preparation techniques; bottom-up proteomics; suspension trap; filter-aided sample preparation; sodium dodecyl sulfate; label-free quantification; digestion comparison; tandem mass spectrometry; quantitative proteomics


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Introduction Global identification and quantification of proteins and peptides has become a critical tool for the analysis of biological systems1,2,3. Mass spectrometry-based studies using ultra performance liquid chromatography (UPLC)4,5,6 or capillary zone electrophoresis (CZE)7,8,9 coupled to a mass spectrometer are often performed to identify thousands of proteins in a sample of interest. Large-scale proteomic studies often rely on digestion of proteins into peptides using the enzyme trypsin, and the corresponding peptide ion intensities can be exploited for quantification10. Efficient digestion of proteins into their corresponding peptides is critical for the success of bottom-up protein quantification. The depth of coverage of the proteome is largely dependent on the chosen sample preparation and separation techniques. Sample preparation methods upstream of mass spectrometry can vary greatly depending on sample type, lysis conditions, digestion, and offline fractionation methods, with each method having distinct advantages and drawbacks. A number of previous studies have compared lysis conditions11,12, fractionation methods13,14, and digestion conditions10,15,16 to derive the optimal proteomic preparation protocol. Most studies emphasize the importance of detergents to be able to detect hydrophobic membrane proteins that perform many important functions in the cell. The most commonly used detergent to achieve this result is sodium dodecyl sulfate (SDS), which readily solubilizes proteins in biological matrices. However, SDS removal is critical before mass spectrometry analysis due to its ability to contaminate liquid chromatography systems and dominate mass spectra. The most popular method for detergent removal and digestion in recent years has been filter-aided sample


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preparation (FASP)17, which was designed based on previous methods from Manza et al. using spin filters18. In this preparation, SDS-containing protein samples are applied to a filter and washed with 8M urea to disrupt SDS micelles. Filters are then washed with various buffers to remove excess urea before digestion. Digestion is performed on the filter and peptides are eluted the following day. Although this method has been widely successful for many applications19,20,21, the tedious nature of the protocol and batch-tobatch variation often hinders its use for high-throughput proteomic studies. For these reasons, new technologies have been developed to aid SDS-based proteomic preparations. Recently, a suspension trapping (S-Trap) method was described by Zougman et al. which allows for preparation of SDS-containing protein lysates in a fraction of the time needed for FASP protocols22. In this method, proteins are lysed in 5% SDS and a fine protein particulate suspension is created through the addition of phosphoric acid and a methanolic buffer solution. The suspension is trapped in a stack of filtration material and residual SDS is washed away in one short wash step. Proteins are digested in the filter using a protease of choice before analysis via LC-MS/MS. The STrap possesses the advantages of FASP and other filter-based methods while decreasing the sample handling steps and time prior to mass spectrometry analysis. In this study, we examined the quantitative and qualitative differences in proteins from the SW480 colon cancer cell line that were lysed and digested under various conditions. We compare commonly used SDS and urea-based lysis buffers, and digest these lysates using a traditional in-solution approach, a FASP method, and an S-Trap method. We used label-free quantification in MaxQuant23 to determine quantitative


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differences that occur due to the various lysis and digestion strategies. The results of this work show that each method of digestion preferentially enriches different parts of the proteome and yields different total numbers of protein identifications. Furthermore, we found that filter-based methods were more consistent across experimental replicates when compared to in-solution digests. It is critical to assess the efficacy and robustness of these proteomic methods to determine their use in bottom-up proteomic studies with clinical and biological consequences. Materials and Methods Reagents Cell culture reagents and phosphate buffered saline were purchased from Invitrogen (Gaithersburg, MD). Fetal bovine serum (FBS) was obtained from Hyclone. Sodium dodecyl sulfate (SDS), triethylammonium bicarbonate (TEAB), urea, iodoacetamide (IAA), dithiothreitol (DTT), sodium orthovanadate, were purchased from Sigma Aldrich (St. Louis, MO). TPCK-treated trypsin was obtained in-house. Mass spectrometry solvents were obtained from Burdick and Jackson (Muskegon, MI). Suspension-traps were purchased from Protifi (Huntington, NY), while FASP filters were obtained from Millipore (Burlington, MA). Cell Culture and Protein Harvest Colorectal cancer SW480 cells were purchased from the American Type Culture collection (ATCC) and cultured in RPMI medium supplemented with 10% FBS and Lglutamine. Cells were maintained at 37oC with 5% CO2 and were verified by STR sequencing in the summer of 2016. Cells were grown to 80% confluency and harvested with one of two lysis buffers. The first lysis buffer contained 8M urea, 75 mM NaCl, 50


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mM Tris-HCl (pH 8), 1 mM NaF, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM PMSF, and 1 tablet of EDTA-free protease inhibitor cocktail. The second lysis buffer contained 3% SDS in 50mM Tris-HCl (pH 8), 1 mM NaF, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, and 1 tablet of EDTA- free protease inhibitor cocktail. Cells were harvested in 2mL of lysis buffer and sonicated on ice for three one-minute rounds at 15% amplitude. SDS lysates were heated to 90oC for 10 minutes. Lysates were then clarified at 15,000 rpm for 10 minutes. Total protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce, Rockford, IL). 100µg aliquots of sample were used for all subsequent steps. In-solution Sample Preparation Only proteins harvested with the 8M urea buffer were prepared using in-solution methods. 100µg of proteins was reduced with 5mM DTT for 1 hour at 37oC. Proteins were then alkylated with 14mM IAA for 30 minutes in the dark. The alkylation reaction was quenched with an additional 5mM DTT at room temperature for 1 hour. Samples were diluted with 50mM Tris-HCl so that the final concentration of urea was 1.5M. Trypsin was added 1:50 (enzyme:protein) and digested overnight at 37 OC. Digestion reactions were quenched with the addition of 10% formic acid until the pH 2 or log2

Comparison of In-Solution, FASP, and S-Trap Based Digestion

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