Proteomic Sample Preparation through Extraction by Unspecific

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Proteomic sample preparation through extraction by unspecific adsorption on silica beads for ArgC-like digestion Yannik Lewin, Moritz Neupärtl, Vahid Golghalyani, and Michael Karas J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00882 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Journal of Proteome Research

Proteomic sample preparation through extraction by unspecific adsorption on silica beads for ArgC-like digestion Yannik Lewin1‡, Moritz Neupärtl1‡, Vahid Golghalyani1,2* and Michael Karas1† 1

Institute of Pharmaceutical Chemistry, Goethe-University, Frankfurt am Main 60438, Germany

2

Biopharmaceutical Development, Analytical Sciences. MedImmune Ltd, Granta Park, Great Abington, CB21 6GH, United Kingdom Mailto: [email protected]

‡

Authors contributed equally

*

Author to whom correspondence should be addressed: +44 20 374 96358

1 ACS Paragon Plus Environment

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Abstract Sample preparation for mass-spectrometry-based proteomic analyses usually requires intricate, multistep workflows that are often limited in capacity or suffer from sample loss. Here, we introduce a lean adsorption-based protocol (ABP) for the extraction of proteins from fresh cell lysates that enables us to modify and tag protein samples under harsh conditions, such as organic solvents, high salt concentrations or low pH values. This offers high versatility while also reducing the required steps in the preparation process significantly. Protein identifications are slightly increased compared to traditional acetone precipitation followed by an in-solution digestion (AP/IS) or Filter Aided Sample Preparation (FASP) and proved complementary to both methods regarding proteome coverage. When combined with ArgC-like digestion this approach delivered 5386 uniquely identified proteins, a substantial increase of 18.27% over tryptic digestion (4554), while decreasing spectra complexity due to a lower number of peptide to spectra matches per protein and the number of missed cleaved peptides. In addition, an increased number of identified membrane proteins and histones as well as improved fragmentation and intensity coverage were observed through comprehensive data analysis.

Keywords: chemical modification, proteomic sample preparation, ArgC-like digestion, protein enrichment


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1 Introduction The most common procedure in mass spectrometry (MS) based proteomics is referred to as bottom-up proteomics and relies on proteomic samples being extracted, purified and digested into peptides by sequence-specific proteases during sample preparation before being separated by liquid chromatography (LC) and subjected to electrospray ionization (ESI) and tandem MS (MS/MS). Sample preparation presents a crucial step in this workflow due to its impact on robustness, accuracy and sensitivity of the analysis1 since it encompasses cell lysis, protein extraction, enrichment, purification, denaturation, chemical modification and enzymatic digestion. Methods in this particular field have been described in protocols relying on protein precipitation, such as acetone precipitation

2,3

or

suspension trapping (STrap)4. Other well established approaches, like the popular filter aided sample preparation (FASP)5,6, minimal, encapsulated proteomic-sample processing (iST)7 or single-pot solid-phase-enhanced sample preparation (SP3)8, have been evaluated and compared recently by Silaf et al9. FASP, as first described by Manza et al.5 and its proximate full realization by Wisniewski et al.6, changed the paradigm of sample preparation by introducing ultrafiltration membranes to remove surfactants, small molecules and salts from the sample through molecular weight cut off filters. Described downsides are long workflows and limited initial protein amount. SP3 binds proteins to paramagnetic carboxylate beads with organic solvents through mechanisms of hydrophilicinteraction chromatography (HILIC) and electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)10,11 and elution is carried out with aqueous solutions. ArgC-like digestion using trypsin requires propionylation of lysines (K), causing an inhibition of one of the two specific cleavage sites of trypsin. Originally, the chemical modification of proteins for ArgC-like digestion depended on covalent immobilization of protein samples onto magnetic beads featuring N-Hydroxysuccinimid(NHS) groups, which rendered exchange of aqueous and organic solvents possible.12 Covalent binding however not only demands an additional reaction step during sample preparation, it also removes the bound peptides from the analysis. Therefore, a switch to non-covalent immobilization is favorable and can be performed with silica beads. The products of this process are peptides equivalent to the ones generated by the protease ArgC, yet devoid of byproducts caused by the lacking specificity associated with this enzyme.13 At the same time the cleavage efficiency of trypsin is increased leading to a strong reduction of peptides with missed cleavages. Overall, ArgClike digestion leads to a higher number of identified proteins and a shift towards higher ion scores that result in a higher proteome coverage, supported by the higher cleavage specificity and fragmentation efficiency of the



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resulting Arg-C-like peptides.12,14 Aside from qualitative improvements, ArgC-like digestion has recently been described as a method for quantification by Schräder et al15. In general, adsorption of protein samples on various surfaces is a well-known issue and is mostly regarded as an unwanted cause for sample loss in vials, pipet tips and well plates16,17. By turning the tables and utilizing adsorption as a means for extraction, we created an adsorption-based protocol (ABP) relying on paramagnetic silica particles low in diameter and large in surface. The mechanism behind ABP is well documented as protein adsorption on silica surfaces and includes the effects of pH, salt concentrations and presence of pores within the surface18–23. Non-magnetic borosilicate particles have been used for DNA purification24 with high salt concentrations already, silica particles for DNA extractions from gels can be acquired from Thermo Fisher Scientific. In the first part of this report the performance of this simple method will be displayed, along with its versatility and robustness. The optimized protocol is then applied to fresh whole-cell lysates in direct comparison to FASP and acetone precipitation/in-solution digestion AP/IS, examining the relative performance regarding identifications, preparation time, reproducibility and potential complementarity using tryptic digestion to establish it as a tool for sample preparation in its own right. After validating ABP as a stand-alone method of equal or better performance compared to FASP and AP/IS, the second part of the report presents the combination of ABP with the advantages provided by ArgC-like digestion to demonstrate the applicability of ArgC-like digestion and to discuss its many benefits over conventional tryptic digestion.


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2 Materials and Methods 2.1 Sample Preparation Human apo-serotransferrin, bovine serum albumin (Sigma Aldrich, Steinheim, Germany) and Escherichia coli cell lysate (BioRad, Hercules, CA, USA) were dissolved, depending on the protocol, in either 0.1M triethylammonium-bicarbonate buffer (TEAB) (Sigma Aldrich), phosphate buffer pH 7.4 or 3.0. HEK 293T cells, kindly provided by working group of Prof. Dr. R. Marschalek (institute for pharmaceutical biology, Goethe university of Frankfurt, Germany), were cultured in HyClone (DMEM/high glucose) medium supplemented with bovine calf serum (both GE Healthcare, South Logan, USA and Pasching, Austria), glutamine (Sigma Aldrich) and penicillin/streptomycin (Biochrom GmbH, Berlin, Germany). Cells were harvested after removal of medium, washed with Dulbecco phosphate buffered saline (PBS) (GE Healthcare, South Logan, USA), rinsed from the petri dishes with the latter and carefully centrifuged to a pellet for 5 min at 80 g. After removal of supernatant the cell pellet was weighted and lysed with 500µL urea 8M and 3µL Lysonase (Merck Millipore, Burlington, USA) per 100mg cell pellet. After centrifugation, the pellet of cellular debris was discarded and the supernatant collected followed by evaluation of the approximate protein content via BCA assay (Thermo Scientific, Rockford, USA)25. Magnetic NHS- and aldehyde-activated silica beads (Bioclone Inc., San Diego, USA) were activated by suspension in cold 1mM HCl. Plain silica beads (Bioclone Inc.) only required suspension in the incubation buffer followed by a washing step in the same. Immobilization of the proteins on magnetic beads was done according to protocol provided by Bioclone. Incubation of NHS beads was quenched by replacing the incubation buffer with 160mM ammonium-bicarbonate buffer (Sigma Aldrich), while for the silica beads the incubation buffer was replaced with TEAB 100mM. Filter aided sample preparation (FASP) was done with filters from and according to protocol by Expedeon (Swavesey, UK). Acetone precipitation was achieved by adding a surplus of ice-cold acetone to the cell extract ten to one and leaving the proteins precipitate over the course of at least 4h at -20°C. After pelletizing the protein at 18k g and drying, the pellet was dissolved in TEAB 100mM for modification and digestion. Compositions and concentrations are described in the supplementary table S1, protocols by manufacturers are provided in the supporting information.



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2.2 Protein Quantification BCA assay was obtained from Thermo Fisher. For cell lysates, the assay was carried out with 10µL of lysate according to the manufacturer’s protocol against the included external standard of BSA in a range of 1252000µg/mL. Samples containing commercially available Escherichia coli lysate (BioRad) were measured against a standard of the same lysate (BioRad). Relative extraction performance of the paramagnetic beads was determined with an adapted BCA protocol. Blank samples of silica beads to account for the reductive potential of the beads (blanks) and protein immobilized on beads were incubated (30min, 37°C) in a volume of 1mL BCA solution after a repeated washing step with phosphate buffer (pH 3). 1mL of BCA solution was mixed with protein solution from before extraction. Flat bottom 96 well plates were used for this assay and analyzed with a TECAN infinite M200 3 flashes at a wavelength of 562nm (bandwidth 9nm) after shaking for 5s. Absorption measured for the blank samples was subtracted from samples containing beads and protein. The quotient of protein on beads adjusted by blanks and the absorption measured for the pure protein solution describes the relative extraction performance. Cell lysate was analyzed via SDS-PAGE on 12% polyacrylamide gel with an 8% stacking gel (composition as described in sup. table S1) under reductive conditions. Gels were developed including Precision Plus Protein Dual Xtra (BioRad) as a protein standard at a constant voltage of 100V for 80 minutes in a PROTEAN electrophoresis chamber (BioRad). Gels were stained with Coomassie blue (sup. table S1).


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2.3 Protein Modification 100µg bead-bound proteins were reduced with 5µL dithiothreitol (DTT) solution (30mg/mL, Sigma Aldrich) for 45 min at 57°C and carbamidomethylated with 5µL iodoacetamide (IAA) solution (65mg/mL, Carl Roth GmbH, Karlsruhe, Germany) over 45min at room temperature in the dark. Buffer removal and washing terminates the modification. Reduced and alkylated samples for ArgC-like digestion were washed once with TEAB for removal of potentially present ammonia before being suspended in acetonitrile (ACN) and derivatized with 1µl/100µl suspension of propionic acid anhydride (PA, Sigma Aldrich) for 2h at 4°C.

2.4 Digestion Beads were washed twice with TEAB 100mM and transferred to a fresh vial before being suspended in 90µL TEAB 100mM and 10µL of 1mM HCl containing 1µg Trypsin. For all approaches digestion was stopped after shaking for 18h at 37°C by adding 8-10µL trifluoroacetic acid (10%) until a pH of 3 was reached. The peptide samples were purified and enriched using self-packed StageTips as described by Rappsilber et al.26

2.5 Mass Spectrometry LC-MS/MS measurements for comparison with FASP were carried out on a Q Exactive coupled with a Dionex Ultimate 3000 LC unit and a Nanospray Flex Ion-Source (all Thermo Scientific). HPLC peptide separation was carried out by C18 reverse phase (RP) precolumn (Thermo Scientific) and by a picofrit emitter tip (New Objective, diameter 100 μm, length 15 cm) packed in-house with Reprosil C18 resin 2.4µm (Dr. Maisch, Germany). A gradient of 5% mobile phase A (4% acetonitrile, 0.1% formic acid) to 30% mobile phase B (80% acetonitrile, 0.1% formic acid) for 70min at 300nL/min flow rate was applied. A second gradient to 60% B for 30min with a flow rate of 300nL/min precedes the final 99% B for 5min and re-equilibration of the columns with 1% B. MS spectra were recorded in positive ion mode with a scan range of 300 to 2000m/z (MS, MS/MS), a resolution (FWHM) of 70 000 (MS) and 17 500 (MS/MS). Automatic gain control (AGC) values were set to 3×106 (MS) and 105 (MS/MS) total ion counts. Maximal ion injection time of 160 ms (MS) and 150 ms (MS/MS) was selected. 15 ions of highest abundance and with positive charges (2+ or higher) were selected for MS/MS scans with an isolation window of 2m/z and excluded from precursor selection 30s after fragmentation. For data acquisition XCalibur software (Thermo Scientific, Waltham, USA) was used.



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Samples for ArgC-like digestions were analyzed on a Q-Exactive Plus paired with an EASY 1000 nLC. A column of 75µm I.D. x 500mm was packed with C18 resin (2.7µm, Poroshell EC120, Agilent, USA) for peptide separation. Samples were analyzed using a 4h gradient of solvent A (0.1% formic acid in water) to 50% solvent B (0.1% formic acid in 80% acetonitrile) at a flow rate of 250nL/min. Peptide loading was performed with solvent A. MS scans were acquired at a resolution of 70 000, followed by 10 MS/MS scans at a resolution of 17 500 (AGC 5×10E5, max. ion injection time of 55 ms, isolation window 1.8m/z).

2.6 Data Analysis File Processing. LC-Tandem-MS files from Thermo instruments were converted into mzML format with MSConvert27 to conduct Mascot database search. The Python module of the MS Parser package (Matrix Science, London) was used to generate peptide and protein lists extracting the information of the Mascot dat files after the performed database search. The different operations could be stream lined by integrating them into the KNIME Analytics Platform28 (including database search for the comparison of ABP, AP/IS and FASP). Larger mzML files obtained during the analysis of ArgC-like digestion were handled with Mascot Daemon29. All further data analysis was done in KNIME and is provided in the supplementary information.

Mascot search. SwissProt Database (Ver. 01_2018) search with taxonomy defined as Homo sapiens (20,395 entries) was conducted by using Mascot (vers. 2.6)30 with one of two sets of parameters. For samples with tryptic digestion, carbamidomethylation of cysteines was defined as a fixed, protein N-term acetylation and methionine oxidation as variable modifications. ArgC-like digested samples were defined as having carbamidomethylation of cysteines and propionylation of lysines as fixed modifications. Oxidation of methionines, acetylation and propionylation of protein N-termini were defined as variable modifications and ArgC set as the utilized protease. The search allowed for 2 missed cleavages and a tolerance of 5 ppm for precursor ions and of 0.1 Da MS/MS mass tolerance for fragments. False discovery rates were estimated by using a shuffled decoy database and results were percolated for increased specificity and sensitivity31. Schräder et al. identified the reoccurring signal of 140.107 m/z in MS/MS spectra of ArgC-like digestions as a fragment of propionylated lysine.15 As a consequence, the respective signal was set to be ignored in MS/MS spectra of ArgC-like digestions as not to be falsely defined as an unassigned signal resulting in a biased ion score. Further parameters can be found in supplementary information table S2.


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Figure 1: Protein recovery before and after inactivation of reactive groups on magnetic silica beads (A), effects of potential disruptive agents on adsorption (B)

3 Results 3.1 Robustness of binding capacity Magnetic NHS-Beads originally proved to be an essential tool for ArgC-like digestion as they allow for quick buffer exchange and the usage of organic solvents to optimize derivatization yield. When comparing beads of different diameters and surface reactivity (Bioclone, San Diego, USA) regarding their binding capacity, the amount of unspecific and reversible immobilization was investigated. Reactive surface groups were chemically inactivated before protein incubation. Quenching of the reactive groups was done with the blocking solutions according to the manufacturer’s manual (sup. table S1) and the performances of active and inactive beads was compared. When choosing particles of 1µm size and thus high specific surface, the amount of unspecifically immobilized protein far outweighed the one covalently bound, making NHS groups potentially superfluous for enrichment on silica beads. In attempts to make optimal use of this effect, pH dependency was investigated. Escherichia coli lysate was chosen as a diverse mixture of proteins. Figure 1A shows protein recovery on magnetic beads with a silica surface featuring reactive groups for covalent immobilization. The performance remained unaltered after inactivation of the beads’ reactive functional groups leaving only unspecific, non-covalent interaction as means for immobilization. Unmodified silica beads immobilized up to 95% of protein from Escherichia coli lysate on silica bead surface at pH 4 (Figure 1B). To evaluate robustness, 20µg Escherichia Coli lysate was dissolved in 100µL of various solvents



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and kept at the previously established pH. Proteins were extracted with magnetic silica beads, washed and analyzed via BCA assay. The presence of 500mM NaCl, 50% Methanol, 20% Glycerol, 200mM DTT or 10% noctylglucoside showed no significant negative effects. The addition of a chaotropic agent like urea might increase protein solubilization32 therefore slightly decrease adsorption. Glycerol and n-octylglucoside showed a minor positive effect on the extraction process. However, the immobilization was inhibited by 10% SDS, which reduced protein recovery down to 5%. When diluting the samples from previously 100µL to 1mL (0.02mg/mL protein concentration) with additional buffer, recovery in the presence of now only 1% SDS improved slightly but remained unsatisfactory. In spite of the sample dilution, the extraction performance across the remaining samples only decreased mildly and even improved in case of urea. (Figure 1B). To further evaluate extraction performance, 20µg of protein from HEK 293T cell lysate was extracted with magnetic beads at pH 4, reduced, alkylated and digested with trypsin subsequently. Equivalents of 20µg protein for lysate, supernatant after extraction and after digestion were loaded onto a polyacrylamide gel (12%) and gel electrophoresis was conducted. The extraction of proteins from the cell lysate and subsequent desorption of tryptic peptides after on-bead digestion are visible after staining with Coomassie blue (supplementary figure S1). With a satisfying extraction performance in the absence of SDS, unmodified silica beads were chosen for further evaluation and eventually utilized in the improved sample preparation protocol for ArgC-like digestion (Figure 2).

Figure 2: General workflow of ArgC-like digestion from lysis to ArgC-like peptides in one vial.


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3.2 Performance of beads compared to FASP and in-solution digestion Based on the robustness observed and demonstrated before, the potential of ABP for direct extraction of protein from fresh, detergent-free cell lysates was recognized. A comparison of ABP to other approaches commonly used in proteomic sample preparation (for which FASP and in-solution digestion were chosen) therefore is a logical progression to further evaluate the adsorption-based protocol. For this, HEK 293T cells were lysed with urea 8M and the protein amount was determined via BCA assay as described in methods and materials. Triplicates for each protocol were prepared using volumes equivalent to 100µg protein. The proteome samples were incubated with 10µL magnetic silica beads (40mg/mL) together with phosphate buffer at a final pH of 4 and reduced and alkylated prior to digestion. Parallel to the on-bead digestion, samples equal to 100µg of protein of the same lysate were either precipitated with ice cold acetone or transferred onto a size-exclusion membrane for FASP. To achieve comparability, all three protocols of sample preparation were carried out with the same fresh cell lysate in the same timeframe, reduced with DTT, carbamidomethylated with IAA and digested with trypsin overnight at 37°C. (Alternatively simultaneous reduction and alkylation using TCEP and ClAA, as described by Kulak et al.7, can be performed.) The proteins identified are results of a Mascot database search with the parameters described in methods and materials. Only peptides with a percolator adjusted ion score above an identity threshold of 13 were employed for protein identification. Table 1 displays the sum of identified peptides and corresponding proteins including their calculated false discovery rates (FDR). The generally low FDRs for peptides across all protocols ranked from low to high beginning with the bead-based approach (0.14%), followed by FASP (0.17%) and insolution digestion (0.22%). Figure 3A shows the examination of the listed proteins as Venn diagrams comparing the number of identified proteins including the ones unique to each method of preparation. 3139 proteins were discovered in total, while the identifications shared by all three methods (1600) only make up little more than half of that. The complementarity of the methods therefore is significant and e.g. increases the number of identifications by 30% over solely using FASP. Both, the in-solution method and the bead-based approach yielded a 10.53% and 10.16% increase in identifications respectively. For peptides the numbers vary even further, with the ABP (10715) producing 19.99% more uniquely identified peptides over FASP (8930) and 10.10% more than the precipitated sample (9732).



Protocol

Uniq. peptides

Uniq. proteins

FDR peptides

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MC 1

MC 2

ABP 10715 2417 0.14% 25.21% 5.06% FASP 8930 2194 0.17% 18.38% 2.31% AP/IS 9732 2425 0.22% 24.54% 4.79% Table 1: Identified peptides and respective proteins in total for adsorption-based protocol (ABP), FASP and acetone precipitation/in solution digestion (AP/IS) including false discovery rates generated by a decoy database derived from three replicates. Last two columns represent the relative amount of one or two missed cleaved (MC) peptides, respectively. (see sup. tables S3a-d for absolute numbers and replicates)

To exclude any potential bias towards hydrophobicity, acidity or size further analysis regarding gravy score, isoelectric point and length in number of amino acids was conducted. No preferable treatment towards either of these parameters could be detected. For all three approaches the distributions, included in supplementary data figures S2 and S3, appear almost identical. The median is close to -0.4 on the GRAVY scale, while 50% of the identified proteins show a score between -0.3 and -0.7, placing the clear majority of identifications in the hydrophilic spectrum. Similar results regarding the isoelectric point (IP), where 50% of the identified proteins show an IP between 5.5 and 8.5, the median off-center at 6.5. The distributions in protein size appear equally similar, where no size exclusion effect can be observed. To investigate the complementarity of the three methods we analyzed the proteins exclusive to each protocol separately. No significant differences were observed, neither on protein level, nor on level of identified peptides. However, the ABP shows a cut-off effect at peptides longer than 60 amino acids, hypothetically due to continued adsorption onto the silica surface. An increase in methionine oxidation was observed for ABP, where an average of 70% of all methionine containing peptides carried at least one oxidation (sup. figure S4, table S4). Compared to the average 26.75% (AP-IS) and 30.14% (FASP) this presents a significant shift, most likely catalyzed by the presence of iron in magnetic silica beads33. Since methionine oxidation is usually considered in the database search, this effect might be beneficial by further denaturing the protein and thus promoting the adsorption process. Additionally, we compared the composition of peptides exclusive to each protocol. Regarding occurrences of acidic, basic, hydrophobic and hydrophilic amino acids as well as the overall peptide length we found that ABP and the AP-IS approach show no difference except for the before mentioned cut-off effect at 60 amino acids for ABP. Peptides exclusive to FASP appear to be shorter, less hydrophobic and acidic on average (sup. figures S5AE). FASP derived peptides with MCs are less common (Table 1) compared to the other approaches which is especially apparent among the exclusive peptides. Here, 40.03% and 35.03% of peptides exclusive to ABP and AP-IS carry at least one MC, while FASP has a much lower MC rate of only 14.94% (supp. table S3c).


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This could suggest a higher digestion efficiency or a systematic sample loss due to size exclusion of long, missed cleaved peptides during filter aided sample preparation. The reproducibility of each method is described in Figure 3C. The in-solution digestion after acetone precipitation (AP/IS) and the adsorption-based protocol (ABP) show very low standard deviation and therefore high reproducibility in the numbers of identified peptides. The lower reproducibility of FASP compared to a different bead-based approach has already been described by Silaff et al.9 and similar conclusions were drawn by Tanca et al.34 when comparing in-solution digestion with FASP. While the standard deviation among assigned hits is lowest with the in-solution digestion (0.17%), the bead-based approach is very close (0.54%) compared to the high number of 7.78% with FASP (Table 1). In summary, all three methods can most likely be applied equally and complementary for cell lysate analysis without known trade-off or bias according to the presented comparison. The significantly shorter preparation time (figure 3A), low costs per sample yet high numbers in identified peptides and proteins should make magnetic silica beads an attractive tool for proteome research.



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Figure 3: Comparison of the workflow for each method of sample preparation. (A) Venn diagram of identified proteins for each of the protocols (B) Total number of MS/MS spectra, hits above and below the identity threshold and unassigned hits from Mascot queries, reproducibility is described by error bars. (C)


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3.3 Comparison of ArgC-like digestion and tryptic digestion In light of the excellent performance of ABP regarding identifications, reproducibility and practicality as well as the ability to expose proteins to harsh conditions, the potential to perform ArgC-like digestion on silica beads is to be assessed. ArgC-like digestion has been shown to improve proteomic results by providing a shift of distribution towards higher ion scores, proteome coverage and a lower occurrence of peptides with missed cleavages when applied to commercially available Escherichia coli lysates utilizing magnetic NHS-beads for covalent protein immobilization12. To verify the beneficial effects of this alternative digestion method, freshly prepared HEK 293T cell lysates were analyzed while relying on unspecific adsorption method to silica beads (ABP), as established above. In addition, acetonitrile is investigated as a replacement for methanol during propionylation, since alcohols are known for esterification of carboxylic groups like C-termini, aspartic acid (D) or glutamic acid (E)35,36. This risk can be minimized by using high capacity buffers to circumvent the drop to low pH-values caused by propionic acid but can be eliminated entirely by avoiding methanol as a solvent altogether. After cell lysis, immobilization, reduction and alkylation (see materials and methods) one set of triplicates was stored away for tryptic digestion. On top of the routinely performed propionylation in methanol/TEAB buffer 1M (4:1)12, referred to as ArgC-MeOH in the following, a third set of samples was propionylated in 100% acetonitrile (ArgC-ACN) for two hours at 4°C. This is done to eradicate the chance of acidic methylation through methanol, to simplify the protocol and to investigate a potential impact on identifications. A longer LC-MS/MS run was chosen as described in materials and methods with the goal of higher proteome coverage and more comprehensive data on the effects of ArgC-like digestion. Database search for all presented ArgC-like data was performed with Mascot and data processing followed as described in methods (2.6). In a separate database search of the same LC-MS runs, potential carbamylation of lysines, a reported result of high urea concentrations during cell lysis37, was investigated. In this case, carbamylation and propionylation were set as variable modifications of lysines to determine the rate in which both competing modifications occur. The result (sup. table S5) shows a propionylation rate of lysines with a minimum of 99.28% and a maximum of 0.26% of carbamylation in three replicates for peptides above the identity threshold of 13. It is therefore strongly recommended to define propionyl as a fixed modification in ArgC-like digestion as to avoid prolonged database searches and minimize false positive identifications38.



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Figure 4: Sum of all identified proteins as a Venn diagram (A) normed histogram of percolator adjusted ion scores above the identification threshold (B). Reproducibility and MS/MS hits, unassigned and above identity threshold (C), the mean number of peptide spectral matches (PSM) per identified protein (D)


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Table 2 lists uniquely identified peptides and proteins for each protocol including FDRs for peptides. Inherently, ArgC-like digestion produces fewer peptides, since lysines are unavailable as cleavage sites. The substantially higher numbers of identified proteins ArgC-like digestion has been observed and described previously12 and can be confirmed here. False discovery rates are low in all cases and thus affirm the comparability of the results.

Protocol Tryptic ArgC-MeOH ArgC-ACN

Uniq. Peptid.

Uniq. Prot.

FDR Peptides 1 Miss. Cleav.

2. Miss. Cleav

37154 25844 27787

4554 5177 5386

0.22% 0.17% 0.20%

8.93% 1.07% 1.36%

32.30% 13.84% 14.87%

Table 2: Sum of identified peptides and respective proteins derived from three replicates of the tryptic and both ArgC-like digestions including false discovery rates generated by a decoy database and missed cleavages (see supplementary tables S6a-b for absolute numbers)

The number of identifications achieved by the ArgC-like protocols show an increase over the ones derived from tryptic digestion by 18.27% for ArgC-ACN and 13.68% for ArgC-MeOH, respectively. This confirms previously made observations of the advantage of ArgC-like digestion, as reported with an increase by 10% for Escherichia coli lysates by Golghalyani et al.12 A total of 6253 unique proteins have been identified, which is covered by 86.13% from ArgC-ACN derived data, whereas the result of standard tryptic digestion contributes only 72.83% (sup. figure S6). Overlaps in identifications and reproducibility among replicates (supp. table S7) were within normal range for a complex proteomic sample analyzed with LC-Orbitrap instrumentation according to Tabb et al.39. Due to the high similarity of the outcomes by ArgC-ACN and ArgC-MeOH, the focus will rest on ArgCACN for its slightly better overall performance. The Venn diagram of figure 4A visualizes the distribution and overlap of total identifications, between ArgC-ACN and the tryptic protocol and highlights the ones exclusive to each approach. Considering the higher number of identified proteins in the ArgC-like approach, the similar number of hits above identity threshold (figure 4C) in both protocols suggests an average higher number of peptide spectra matches (PSM) originating from the same protein after tryptic digestion. This can be confirmed with a scatterplot (figure 4D): The (x/y) position of each dot is described by the number of mean PSMs per protein, (x) for trypsin-generated peptides, (y) for ArgC-like peptides. Therefore a higher number of PSMs of tryptic peptides originate from highly abundant proteins (larger, darker dots) as estimated via normalized spectral abundance factor (NSAF)40 compared to ArgC-like peptides. This leaves tryptic digestion to be less efficient, as matching a high number of peptides to the same protein convolutes the mass spectra without increasing the overall identifications. ArgC-like digestion in opposition, provides more identifications with decreased spectra complexity. The proteins with the highest PSM per protein



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after ArgC-like digestion were identified as Histones. The effect of ArgC-like digestion on histones is also reflected in their identification coverage and seems to confirm the improved accessibility of histones for mass spectrometry, as described by Garcia et al.41 previously. Filtering of Mascot generated dat files for protein families shows an increase of 53.33% of identified histones compared to the tryptic result (supplementary table S8a). Supplementary table S8b sorts the identified proteins into categories of subcellular structures, which was achieved by integrating all protein related information included in Mascot-generated dat files into the protein identification lists and extracting the data on cellular origin with a dedicated Python script. Here, the identifications of membrane associated proteins displays a disproportionate increase of 28.35% compared to the overall increase after ArgClike digestion (18.27%). Contrary to our previous publication based on an Escherichia coli proteome, a very similar distribution for both protocols regarding the percolator adjusted ion scores was observed when comparing tryptic digestion to the ArgClike approach (figure 4B). Reproducibility was analogous to the comparison of ABP to FASP and AP/IS. The slightly higher number of hits in MS/MS after tryptic digestion in figure 4C is to be expected, due to the higher total number of peptides generated. The hits above identity threshold are similar for both methods in relation to their amount of total hits (ArgC-ACN 34.04%, trypsin 31.29%, sup. table S9). The overall percentage of peptides with one missed cleavage was reduced from 32.30% (trypsin) to 14.87% (ArgC-like) and two missed cleavages from 8.93% to about 1.36% (Table 2). Further analysis of the identified proteins and peptides revealed little difference between tryptic and ArgC-like samples. On protein level, no observable shift in GRAVY score or isoelectric points was found (sup. figure S7). On peptide level, the identified precursors only significantly differ in their distribution of basic amino acids, as ArgC-like peptides inherently carry more lysines on average (sup. figure S8). To conclude the analysis of ArgC-like peptides, fragmentation is analyzed since the reduction in basicity of precursor ions has been linked to improvements in fragmentation behavior by Frese et al14. To characterize the fragmentation behavior of ArgC-like peptides, intensity coverage (sum of the intensities of all identified fragments divided by the sum of total ion signal intensities) was selected, as it provides a comparative value to analyze the fragmentation quality in terms of information per spectrum. Increased peptide fragment intensity coverage was observed with ArgC-ACN and fragmentation yielded cleaner y-ion series by significantly shifting the distribution in favor of y-ion fragments (figure 5A). To determine how different precursor sizes benefit from the improved fragmentation behavior, the precursors (figure 5B) were sorted into five groups of mass ranges for additional


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analysis (sup. table S10). A trend of intensity coverage increasing with the size of the precursors can be observed for both protocols, yet ArgC-like spectra show a higher intensity coverage in all cases (figure 5C). The y-ion fragment ratio appears to be consistent for tryptic precursors across all mass ranges. ArgC-like peptides of smaller size however show much cleaner fragmentation than their tryptic counterparts, yet fragmentation becomes less favorable towards y-ions with increasing precursor size (figure 5D). Considering that over 97% of all precursors fall into the first three mass ranges (sup. table S10), the clear majority of ArgC-like peptides benefit from improved y-ion series.

Figure 5: Overall intensity coverage and y-ion fragment ratio for tryptic and ArgC-like digestion (A), histogram of precursor masses (B), boxplots of intensity coverage (C) and y-ion fragment ratio in separated into mass ranges (D).



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4 Conclusions In the first part we established a reliable, quick and cost-effective new method by using paramagnetic silica beads that improves our sample preparation workflow for standard shotgun proteomics. Extraction, enrichment and chemical modification can be done in one vial with minimal labor. The robustness of a bead-based protocol proved to be high and contrary to the previously mentioned methods, it allows for the usage of organic solvents and chemical modification under water-free circumstances. The identifications are comparable in their quantity as well as their physical and chemical traits. Unlike FASP and acetone precipitation however, surfactants interfere with the described protocol. Considering the high reproducibility, increased number of identified peptides and the much shorter preparation time we believe it to be a valid alternative approach with superior qualities whenever surfactant is absent or modifications under harsh conditions are required. Reduction of protein samples at 57°C, methionine oxidation and carbamidomethylation of cysteines are common during sample preparation and inherently lend themselves to ABP by adding to the already occurring protein denaturation during adsorption on silica beads22,42. Additionally these effects could make rigid (“hard”) proteins, which are otherwise less prone to adsorption, available to this method, as the unfolding of proteins increases their surface and conformational entropy43 while decreasing solubility44. The second part not only confirms the applicability of ArgC-like digestion for samples of fresh cell lysates, we were also able to speed up the process by utilizing the adsorption-based protocol (ABP) instead of covalent immobilization. The advantages of ABP in comparison with other protein purification protocols are complemented by the advantages of ArgC-like digestion. These being an overall superior performance regarding increased proteome coverage, lower occurrences of missed cleavages in peptides and higher intensity coverage. Furthermore, we observed cleaner fragmentation in favor of y-ion series and a lower overabundance of PSM per identified protein. The additional disproportionately higher identifications among membrane proteins and histones should be of interest in their respective fields. We are convinced that this approach is not only a valid alternative but a substantial improvement over tryptic digestion in higher microgram ranges.


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Supporting Information The following supporting information is available free of charge at ACS website http://pubs.acs.org

Supporting tables (SuppTables.xlsx): Table S1

Reagents, buffers and solutions prepared in-house

Table S2

Mascot Search parameters

Table S3

Missed cleavages and False discovery rates (comparison ABP, AP/IS, FASP)

S3a

IDs and FDRs

S3b

MCs (all peptides)

S3b

MCs (exclusive peptides)

S3d

Protein ID overlaps

Table S4

Methionine oxidation rate

Table S5

Carbamylation rate of lysines

Table S6

Missed cleavages and False discovery rates (comparison Tryptic and ArgC-like)

S6a

MCs

S6b

IDs and FDRs

Table S7

Protein ID overlaps

Table S8

Protein Origins

S8a

Histones

S8b

Cellular substructures

Table S9

Hits above identity threshold

Table S10

Precursor mass ranges



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Supporting figures (in Supp. Information.docx) Figure S1

Extraction performance of silica beads analyzed with SDS PAGE

Figure S2

Comparison of distributions regarding GRAVY score and isoelectric points

Figure S3

Distribution of peptide lengths of identified proteins

Figure S4

Oxidation rate of methionines

Figure S5

Physicochemical qualities of exclusive peptides

Figure S6

Overlaps of identified proteins

Figure S7

Comparison of GRAVY scores and IPs

Figure S8

Physicochemical qualities of tryptic and ArgC-like peptides

Figure S9

Database search (KNIME workflow)

Figure S10

Determination of subcellular location and histone count (KNIME workflow)

Supporting material (in Supp. Information.docx) Protocols provided by manufacturers


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Acknowledgements 

CECAD/CMMC Proteomics Core Facility, University of Cologne, Cologne 50931, Germany



Working Group R. Marschalek, Institute of Pharmaceutical Biology, Goethe-University, Frankfurt 60590, Germany



Ilka Wittig, Functional Proteomics, Centre for Biochemistry, Med. School, Goethe-University, Frankfurt 60590, Germany



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For TOC only


Proteomic Sample Preparation through Extraction by Unspecific

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