Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid Extraction

The main challenge of bottom-up proteomic sample preparation is to extract proteomes in a manner that enables efficient protein digestion for subsequent mass spectrometric analysis. Today's sample preparation strategies are commonly conceptualized around the removal of detergents, which are essential for extraction but strongly interfere with digestion and LC-MS. These multi-step preparations contribute to a lack of reproducibility as they are prone to losses, biases and contaminations, while being time-consuming and labor-intensive. We report a detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which consists of three mandatory steps, acidification, neutralization and digestion. SPEED is a universal method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction by complete sample dissolution. The protocol is highly reproducible, virtually loss-less, enables very rapid sample processing and is superior to the detergent/chaotropic agent-based methods FASP, ISD-Urea and SP3 for quantitative proteomics. SPEED holds the potential to dramatically simplify and standardize sample preparation while improving the depth of proteome coverage especially for challenging samples. The main challenge of bottom-up proteomic sample preparation is to extract proteomes in a manner that enables efficient protein digestion for subsequent mass spectrometric analysis. Today's sample preparation strategies are commonly conceptualized around the removal of detergents, which are essential for extraction but strongly interfere with digestion and LC-MS. These multi-step preparations contribute to a lack of reproducibility as they are prone to losses, biases and contaminations, while being time-consuming and labor-intensive. We report a detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which consists of three mandatory steps, acidification, neutralization and digestion. SPEED is a universal method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction by complete sample dissolution. The protocol is highly reproducible, virtually loss-less, enables very rapid sample processing and is superior to the detergent/chaotropic agent-based methods FASP, ISD-Urea and SP3 for quantitative proteomics. SPEED holds the potential to dramatically simplify and standardize sample preparation while improving the depth of proteome coverage especially for challenging samples. The majority of mass spectrometry-based proteome studies are currently performed using a bottom-up approach, which relies on the digestion of proteins into smaller peptides (1Leitner A. Aebersold R. SnapShot: mass spectrometry for protein and proteome analyses.Cell. 2013; 154: 252-252 e251Abstract Full Text PDF PubMed Scopus (13) Google Scholar). Different sample preparation methods have been developed aiming at enabling comprehensive and reproducible generation of peptides from proteomes extracted from a large variety of sample types. Current protocols employ detergents, e.g. sodium dodecyl sulfate (SDS) 1The abbreviations used are:SDCsodium deoxycholateCAA2-ChloroacetamideDTTDithiothreitolFASPfilter-aided sample preparationFDRfalse discovery rateHCDhigher-energy c-trap dissociationIAAiodoacetamideISDin-solution digestioniSTin-stagetip sample preparationNCEnormalized collision energyPSMpeptide spectrum matchSP3single-pot solid-phase-enhanced sample preparationSPEEDsample preparation by easy extraction and digestionStrapsuspension trappingTCEPtris(2-carboxyethyl)phosphineTHSDTukey's honestly significant difference. 1The abbreviations used are:SDCsodium deoxycholateCAA2-ChloroacetamideDTTDithiothreitolFASPfilter-aided sample preparationFDRfalse discovery rateHCDhigher-energy c-trap dissociationIAAiodoacetamideISDin-solution digestioniSTin-stagetip sample preparationNCEnormalized collision energyPSMpeptide spectrum matchSP3single-pot solid-phase-enhanced sample preparationSPEEDsample preparation by easy extraction and digestionStrapsuspension trappingTCEPtris(2-carboxyethyl)phosphineTHSDTukey's honestly significant difference., or chaotropic agents, such as urea, for protein extraction and support sample lysis by physical disruption methods, such as heat, ultra-sonication or grinding (2Leon I.R. Schwammle V. Jensen O.N. Sprenger R.R. Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis.Mol. Cell. Proteomics. 2013; 12: 2992-3005Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 3Sielaff M. Kuharev J. Bohn T. Hahlbrock J. Bopp T. Tenzer S. Distler U. Evaluation of FASP, SP3, and iST protocols for proteomic sample preparation in the low microgram range.J. Proteome Res. 2017; 16: 4060-4072Crossref PubMed Scopus (132) Google Scholar, 4Tanca A. Biosa G. Pagnozzi D. Addis M.F. Uzzau S. Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome.Proteomics. 2013; 13: 2597-2607Crossref PubMed Scopus (82) Google Scholar). As many extraction reagents inhibit enzymatic digestion of proteins and are incompatible with LC-MS/MS, the idea behind most sample preparation methods is to remove interfering substances before digestion either by filtration (FASP) (5Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5043) Google Scholar), precipitation (on-pellet digestion, STrap) (6Zougman A. Selby P.J. Banks R.E. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis.Proteomics. 2014; 14 (1006–0. Epub 2014 Mar 26)Crossref PubMed Scopus (173) Google Scholar, 7Duan X. Young R. Straubinger R.M. Page B. Cao J. Wang H. Yu H. Canty J.M. Qu J. A straightforward and highly efficient precipitation/on-pellet digestion procedure coupled with a long gradient nano-LC separation and Orbitrap mass spectrometry for label-free expression profiling of the swine heart mitochondrial proteome.J. Proteome Res. 2009; 8: 2838-2850Crossref PubMed Scopus (109) Google Scholar) or bead-based purification (SP3) (8Hughes C.S. Foehr S. Garfield D.A. Furlong E.E. Steinmetz L.M. Krijgsveld J. Ultrasensitive proteome analysis using paramagnetic bead technology.Mol. Syst. Biol. 2014; 10: 757Crossref PubMed Scopus (513) Google Scholar). However, enhancing lysis by use of physical disruption methods and subsequent protein purification requires additional sample handling steps, which are associated with attendant losses, biases and possible contaminations, while being time-consuming and labor-intensive. sodium deoxycholate 2-Chloroacetamide Dithiothreitol filter-aided sample preparation false discovery rate higher-energy c-trap dissociation iodoacetamide in-solution digestion in-stagetip sample preparation normalized collision energy peptide spectrum match single-pot solid-phase-enhanced sample preparation sample preparation by easy extraction and digestion suspension trapping tris(2-carboxyethyl)phosphine Tukey's honestly significant difference. sodium deoxycholate 2-Chloroacetamide Dithiothreitol filter-aided sample preparation false discovery rate higher-energy c-trap dissociation iodoacetamide in-solution digestion in-stagetip sample preparation normalized collision energy peptide spectrum match single-pot solid-phase-enhanced sample preparation sample preparation by easy extraction and digestion suspension trapping tris(2-carboxyethyl)phosphine Tukey's honestly significant difference. The main exception is in-solution digestion (ISD) of proteins, which is based on the extraction of proteins using either digestion-compatible and often acid-labile surfactants or more frequently high urea concentrations and subsequent dilution into a concentration range which is not inhibiting tryptic digestion anymore (9Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates 3rd., J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Crossref PubMed Scopus (2071) Google Scholar, 10Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (991) Google Scholar, 11Chang Y.H. Gregorich Z.R. Chen A.J. Hwang L. Guner H. Yu D. Zhang J. Ge Y. New mass-spectrometry-compatible degradable surfactant for tissue proteomics.J. Proteome Res. 2015; 14: 1587-1599Crossref PubMed Scopus (48) Google Scholar, 12Humphrey S.J. Karayel O. James D.E. Mann M. High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform.Nat. Protoc. 2018; 13: 1897-1916Crossref PubMed Scopus (144) Google Scholar, 13Masuda T. Sugiyama N. Tomita M. Ishihama Y. Microscale phosphoproteome analysis of 10,000 cells from human cancer cell lines.Anal. Chem. 2011; 83: 7698-7703Crossref PubMed Scopus (61) Google Scholar, 14Wang H. Qian W.J. Mottaz H.M. Clauss T.R. Anderson D.J. Moore R.J. Camp 2nd, D.G. Khan A.H. Sforza D.M. Pallavicini M. Smith D.J. Smith R.D. Development and evaluation of a micro- and nanoscale proteomic sample preparation method.J. Proteome Res. 2005; 4: 2397-2403Crossref PubMed Scopus (144) Google Scholar). Urea-based ISD (ISD-Urea) benefits greatly from the reduced number of individual steps needed for sample preparation and is known to be robust, highly reproducible and easy to perform. However, ISD-Urea is not a universal method for bottom-up proteomics as urea contrasts with strong detergents, such as SDS, ineffective for extracting proteins from hard-to-lyse samples, such as tissues or Gram-positive bacteria. Further, urea is known to attach artificial modifications to proteins, known as carbamylations (15Kollipara L. Zahedi R.P. Protein carbamylation: in vivo modification or in vitro artefact?.Proteomics. 2013; 13: 941-944Crossref PubMed Scopus (98) Google Scholar). This prevents samples from being lysed at elevated temperatures, which would be beneficial for proteome extraction especially from challenging samples. The limitations of in-solution digestion arise from the compromise between efficient lysis and low interference with enzymatic activity. In this study we present a new sample preparation method, which overcomes the limitations of ISD but preserves its straight-forward approach. The new method is termed sample preparation by easy extraction and digestion (SPEED) and consists of three mandatory steps, namely acidification, neutralization and digestion. SPEED uses neither detergents nor chaotropic agents for protein extraction but pure trifluoroacetic acid. The method is robust, highly-reproducible, well-suited even for lysis-resistant sample types, inexpensive, requires low hands-on time and can easily be performed by non-experts without the need for special equipment. Tryptic Soy Agar (TSA) ReadyPlatesTM (Merck, Darmstadt, Germany) were inoculated with E. coli K-12 (DSM 3871), S. aureus (DSM 4910) or B. cereus (ATCC® 10987) and incubated at 37 °C overnight. Cells were harvested using an inoculating loop and washed in 2 × 1 ml phosphate-buffered saline (PBS) for 5 min at 4000 × g and 4 °C. Cells were aliquoted, again pelleted and kept at −80 °C until lysis. HeLa cells (ATCC® CCL-2™) were cultivated in DMEM supplemented with 10% FCS and 2 mm l-Glutamine at 37 °C and harvested at 90% confluency by scraping. Cells were washed in 2 × 2 ml phosphate-buffered saline (PBS) for 8 min at 400 × g and 4 °C, aliquoted, again pelleted and kept at −80 °C until lysis. C57BL/6 mouse lung and liver were obtained from an animal of the central experimental animal facility (MF3, Robert Koch Institute, Berlin, Germany) which was killed in experiments approved by the local authority. Tissue samples were washed 3 x by dipping into 10 ml ice-cold PBS, cut into equal slices, aliquoted, flash frozen with liquid nitrogen and kept at −80 °C until lysis. After addition of lysis buffer (FASP, SP3, Urea-ISD) mouse lung samples were transferred to Lysing Matrix D Tubes (MP Biomedicals, Santa Ana, CA) and subjected to grinding. Lyophilized microbiome standard (20 Strain Even Mix Whole Cell Material, ATCC® MSA2002™) was resuspended in 1 ml PBS, divided into 15 aliquots and cells were pelleted for 15 min at 5000 × g and 4 °C. Samples were resuspended in trifluoroacetic acid (TFA) (Uvasol® for spectroscopy, Merck) (sample/TFA 1:4 (v/v) or 10 μl for the E. coli 1 μg experiment) and incubated at room temperature for 2 (E. coli, B. cereus, S. aureus, HeLa, Microbiome Standard) or 10 (mouse lung) min. B. cereus, S. aureus and microbiome samples were further irradiated for 10 s at 800 W using a microwave oven. Samples were neutralized with 2 m TrisBase using 10 x volume of TFA and further incubated at 95 °C for 5 min after adding Tris(2-carboxyethyl)phosphine (TCEP) to a final concentration of 10 mm and 2-Chloroacetamide (CAA) to a final concentration of 40 mm. Protein concentrations were determined by turbidity measurements at 360 nm (1 AU = 0.79 μg/μl) using GENESYS™ 10S UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and adjusted to 0.25 μg/μl using a 10:1 (v/v) mixture of 2 m TrisBase and TFA and then diluted 1:5 with water. Digestion was carried out for 20 h at 37 °C using Trypsin Gold, Mass Spectrometry Grade (Promega, Fitchburg, WI) at a protein/enzyme ratio of 50:1. A more detailed description of the usage of SPEED is provided as a lab protocol in the supplementary materials. Trifluoroacetic acid is highly corrosive and must be handled using appropriate Personal Protective Equipment!!! HeLa and E. coli cells were prepared using SPEED as previously described except that LC-MS grade TFA and water was used (Thermo Fisher Scientific). Digested samples were acidified using TFA (final concentration 2%) and further filtrated on 200 μl StageTips packed with two MK 360 Micro-Quartz Fiber Filters (MUNKTELL & FILTRAK, Baerenstein, Germany) at 2000 × g for 2 min. HeLa cells were resuspended in trifluoroacetic acid (TFA) (Uvasol® for spectroscopy, Merck) (sample/TFA 1:4 (v/v)) and incubated at room temperature for 2 min. Samples were neutralized with 2 m TrisBase using 10 x volume of TFA and further incubated at 95 °C for 5 min after adding Tris(2-carboxyethyl)phosphine (TCEP) to a final concentration of 10 mm and 2-Chloroacetamide (CAA) to a final concentration of 40 mm. Protein concentrations were determined by turbidity measurements at 360 nm (1 AU = 0.79 μg/μl) using GENESYS™ 10S UV-Vis Spectrophotometer (Thermo Fisher Scientific). 50 μg proteins were diluted to 40 μl using a 10:1 (v/v) mixture of 2 m TrisBase and TFA, mixed with 160 μl acetone and incubated for 2 min at RT. Proteins were captured on Ultrafree®-MC (0.5 ml) centrifugal devices, 0.2 μm, PTFE (Merck) at 5000 × g for 2 min. The samples were washed successively with 200 μl 80% acetone, acetone and n-pentane at 5000 × g for 2 min each. Afterward 40 μl digestion buffer (50 mm ammonium bicarbonate or 1:10 Rapid Digest buffer (Promega)) containing trypsin (1:100 Trypsin Gold, Mass Spectrometry Grade (Promega) or 1:10 Rapid Digestion Trypsin (Promega)) was added and samples were incubated at 37 °C (20h) or 70 °C (15 and 60 min). Peptides were eluted at 5000 × g for 2 min and acidified using formic acid before LC-MS injection. A more detailed description of the usage of fa-SPEED is provided as a lab protocol in the supplementary materials. Samples were suspended in 4% SDS, 100 mm Tris/HCl, 100 mm DTT, pH 7.6 (sample/buffer 1:10 (v/v) or 10 μl for the E. coli 1 μg experiment), incubated at 95 °C for 5 min and further sonicated for 10 (E. coli, HeLa) or 15 (B. cereus, mouse lung tissue) cycles a 30 s at high intensity level and 4 °C using Bioruptor®Plus (Diagenode, Liege, Belgium). Samples were clarified by centrifugation at 16,000 × g for 5 min und processed using Microcon-30kDa Centrifugal Filter Units (Merck) according to the Filter-aided Sample Preparation (FASP) protocol of Wisniewski et al. (5Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5043) Google Scholar) with proteins being digested for 20 h at 37 °C using Trypsin at a protein/enzyme ratio of 50:1. Samples were suspended in 1% SDS, 1x cOmplete Protease Inhibitor Mixture (Roche, Basel, Switzerland), 50 mm HEPES buffer, pH 8.5 (sample/buffer 1:10 (v/v)) or 10 μl for the E. coli 1 μg experiment), incubated at 95 °C for 5 min and further sonicated for 10 (E. coli, HeLa) or 15 (B. cereus, mouse lung tissue) cycles a 30 s at high intensity level and 4 °C using Bioruptor®Plus. Samples were further processed according to the Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) method of Hughes et al. (8Hughes C.S. Foehr S. Garfield D.A. Furlong E.E. Steinmetz L.M. Krijgsveld J. Ultrasensitive proteome analysis using paramagnetic bead technology.Mol. Syst. Biol. 2014; 10: 757Crossref PubMed Scopus (513) Google Scholar), except that proteins were bound to the paramagnetic beads at 70% ACN without acidification as prescribed by Sielaff et al. (3Sielaff M. Kuharev J. Bohn T. Hahlbrock J. Bopp T. Tenzer S. Distler U. Evaluation of FASP, SP3, and iST protocols for proteomic sample preparation in the low microgram range.J. Proteome Res. 2017; 16: 4060-4072Crossref PubMed Scopus (132) Google Scholar). Digestion was carried out for 20 h at 37 °C using Trypsin at a protein/enzyme ratio of 50:1. Samples were suspended in 8 m urea, 50 mm Tris-HCl, 5 mm DTT, pH 8 (sample/buffer 1:10 (v/v) or 10 μl for the E. coli 1 μg experiment) and sonicated for 10 (E. coli, HeLa) or 15 (B. cereus, mouse lung tissue) cycles a 30 s at high intensity level and 4 °C using Bioruptor®Plus. Samples were further incubated for 1 h at 37 °C and clarified by centrifugation at 16,000 × g for 5 min. IAA was added to a final concentration of 15 mm and samples were alkylated for 30 min at room temperature in the dark. Urea was diluted with 50 mm Tris-HCl (pH 8) to 1 m, Trypsin was added at a protein/enzyme ratio of 50:1 and proteins were digested for 20 h at 37 °C. Microbiome standard was prepared using the iST Kit according to manufacturer’s instructions (Preomics, Munich, Germany) in triplicates. Bacterial cell lysis was supported by sonication for 15 cycles a 30 s at high intensity level and 4 °C using Bioruptor®Plus. Microbiome standard was suspended in 5% SDS, 20 mm DTT, 50 mm Tris/HCl buffer, pH 7.6 (sample/buffer 1:10 (v/v)), incubated at 95 °C for 10 min and further sonicated for 15 cycles a 30 s at high intensity level and 4 °C using Bioruptor®Plus. Samples were further processed according to the Suspension trapping (STrap) protocol (6Zougman A. Selby P.J. Banks R.E. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis.Proteomics. 2014; 14 (1006–0. Epub 2014 Mar 26)Crossref PubMed Scopus (173) Google Scholar). Protein concentrations were determined by measuring the tryptophan fluorescence at an emission wavelength of 350 nm using 295 nm for excitation with an Infinite® M1000 PRO microplate reader (Tecan, Maennedorf, Switzerland) (16Wisniewski J.R. Gaugaz F.Z. Fast and sensitive total protein and Peptide assays for proteomic analysis.Anal. Chem. 2015; 87: 4110-4116Crossref PubMed Scopus (159) Google Scholar). The tryptophan content of each sample was determined using a standard curve ranging from 0.1–0.9 μg tryptophan and assuming a tryptophan weight content of 1.3% in the samples. Twenty micrograms protein of each sample type was further digested except for the E. coli experiment with low starting amount (E. coli 1 μg). Therefore, protein content of a cell suspension was determined after TFA-based lysis and cells from the suspension volume equivalent to 1 μg protein were aliquoted and pelleted. The calibration curves for the turbidity measurements (Fig. 5) were determined by dilution of SPEED-derived samples (E. coli, B. cereus, HeLa, Mouse Liver) in 1.7 m TrisBase, 8% TFA, 1.7% SDS. Samples were incubated for 5 min at 95 °C to solubilize proteins. Afterward, fluorescence was measured at an emission wavelength of 350 nm using 295 nm for excitation. A tryptophan standard curve in the range of 0.1–0.9 μg solubilized in 1.7 m TrisBase, 8% TFA, 1.7% SDS was measured in triplicates and was used for protein content determination assuming a tryptophan weight content of 1.3% in the samples. Turbidity of SPEED-lysates or MacFarland standard series (bioMérieux, Marcy-l’Étoile, France) was measured at 360 nm using either a GENESYS™ 10S or an Implen NP80 (Implen, Munich, Germany) UV-Vis Spectrophotometer. If the initial protein concentration was above 1 μg/μl, samples were diluted with a 10:1 (v/v) mixture of 2 m TrisBase and TFA. For experiments analyzing the digestion progress by real-time monitoring, an Infinite® M1000 PRO microplate reader was used. The microplate reader was tempered to 37 °C and turbidity was measured at 360 nm every 5 min. Peptides generated using FASP, SPEED, and Urea-ISD were desalted using 200 μl StageTips packed with three Empore™ SPE Disks C18 (3 m Purification, Inc., Lexington, KY) according to Rappsilber et al. (17Ishihama Y. Rappsilber J. Mann M. Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics.J. Proteome Res. 2006; 5: 988-994Crossref PubMed Scopus (224) Google Scholar) and concentrated using a vacuum concentrator. Samples were resuspended in 20 μl 0.1% formic acid (FA) and peptides were quantified by measuring the absorbance at 280 nm using a Nanodrop 1000 (Thermo Fisher Scientific). Peptide solutions were acidified after SPEED-based processing using TFA and loaded onto six Empore™ SPE Disks C18 packed in 200 μl pipette tips, which were activated using methanol. Peptides were washed with 0.1% TFA, 100 mm ammonium formate (pH = 10) and 20 mm ammonium formate (pH = 10) consecutively. Afterward the peptides were eluted sequentially in 5/7.5/10/12.5/15/17.5/20/50 (v/v) ACN in 20 mm ammonium formate (pH = 10) and dried down using a vacuum concentrator. Samples were resuspended in 20 μl 0.1% FA and peptides were quantified by measuring the absorbance at 280 nm using a Nanodrop 1000. SPEED was used to digest HeLa cells (100 μg) before phosphopeptide enrichment using the high-sensitivity EasyPhos workflow (12Humphrey S.J. Karayel O. James D.E. Mann M. High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform.Nat. Protoc. 2018; 13: 1897-1916Crossref PubMed Scopus (144) Google Scholar). Briefly, 300 μl of the digested samples were mixed with 400 μl isopropanol, 100 μl 48% (v/v) TFA and 8 mm KH2PO4 and 5 mg Titanium dioxide (TiO2) beads (GL Sciences, Tokyo, Japan). Samples were incubated at 40 °C for 5 min and subsequently washed five times using 5% (v/v) TFA/60% (v/v) isopropanol. Beads were removed in a transfer buffer of 0.1% (v/v) TFA/6 0% (v/v) isopropanol using 200 μl StageTips packed with two MK 360 discs. Phosphopeptides were eluted twice in 30 μl 40% ACN and 4% NH4OH and purified using SDB-RPS StageTips (3 m Purification, Inc.) as described elsewhere (12Humphrey S.J. Karayel O. James D.E. Mann M. High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform.Nat. Protoc. 2018; 13: 1897-1916Crossref PubMed Scopus (144) Google Scholar). Peptides were analyzed on an EASY-nanoLC 1200 (Thermo Fisher Scientific) coupled online to a Q Exactive™ Plus mass spectrometer (Thermo Fisher Scientific). 1 μg peptides were separated on a 50 cm Acclaim™ PepMap™ column (75 μm i.d., 100 Å C18, 2 μm; Thermo Fisher Scientific) using a linear 120 (E. coli, B. cereus, high-pH fractions), 180 (HeLa, mouse lung tissue) or 240 (microbiome standard) min gradient of 3 to 28% acetonitrile in 0.1% formic acid at 200 nL/min flow rate. Column temperature was kept at 40 °C using a butterfly heater (Phoenix S&T, Chester, PA). Samples for evaluating online desalting, filter-aided SPEED (fa-SPEED) and phosphopeptide analysis were loaded on a Acclaim™ PepMap™ trap column (20 mm × 75 μm i.d., 100 Å, C18, 3 μm; Thermo Fisher Scientific) at a flow rate of 3 μl/min and were subsequently separated on 200 cm μPAC™ column (PharmaFluidics, Ghent, Belgium) using a 160 min (online desalting) or 210 min gradient (filter-aided SPEED, phosphoproteomics) of 3 to 28% acetonitrile in 0.1% formic acid at 300 nL/min flow rate. Column temperature was kept at 50 °C using a butterfly heater (Phoenix S&T, Chester, PA). The Q Exactive™ Plus was operated in a data-dependent manner in the m/z range of 300–1650. Full scan spectra were recorded with a resolution of 70,000 using an automatic gain control (AGC) target value of 3 × 106 with a maximum injection time of 20 ms. Up to the 10 most intense 2+ - 5+ charged ions were selected for higher-energy c-trap dissociation (HCD) with a normalized collision energy (NCE) of 25%. Fragment spectra were recorded at an isolation width of 2 Th and a resolution of 17,500 at 200 m/z using an AGC target value of 1 × 105 with a maximum injection time of 50 ms. The minimum MS2 target value was set to 1 × 104. Once fragmented, peaks were dynamically excluded from precursor selection for 30 s within a 10 ppm window. Peptides were ionized using electrospray with a stainless-steel emitter, I.D. 30 μm, (Proxeon, Odense, Denmark) at a spray voltage of 2.0 kV and a heated capillary temperature of 275 °C. For the analysis of phosphorylated peptides the maximum injection times were increased to 200 ms (full scan) and 100 ms (MS2) as well as the minimum MS2 target value to 2 × 104. Further, the isolation width was decreased to 1.6 Th and precursor ion charges were restricted to 2–4. All other MS parameters were unaltered. Mass spectra were analyzed using MaxQuant (Version 1.5.1.8 and 1.6.1.0) (18Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar). At first, parent ion masses were recalibrated using the “software lock mass” option before the MS2 spectra were searched using the Andromeda algorithm against sequences from the complete proteomes of either homo sapiens (UP000005640, 92578 sequences, 15/7/16), E. coli K-12 (UP000000625, 4315 sequences, downloaded 26/10/16), B. cereus strain ATCC 10987 (UP000002527, 5821 sequences, downloaded 8/3/17), S. aureus strain NCTC 8325 (UP000008816, 2889 sequences, downloaded 4/10/18) or Mus musculus (UP000000589, 58430 sequences, downloaded 10/1/17), which were downloaded from UniProt. Proteome entries of bacterial species from the microbiome standard were downloaded from UniProt according to their corresponding ATCC® numbers (62989 sequences, downloaded 31/7/18). If no entry was available, the reference proteome of the respective species was used. Spectra were searched with a tolerance of 4.5 ppm in MS1 and 20 ppm in HCD MS2 mode, strict trypsin specificity (KR not P) and allowing up to two missed cleavage sites. Cysteine carbamidomethylation was set as a fixed modification and methionine oxidation as well as N-terminal acetylation of proteins as variable modifications. For phosphopeptide analysis phosphorylation (S,T,Y) was added as an additional variable modifications. The false discovery rate (FDR) was set to 1% for peptide and protein identifications. Dependent peptides with previously unconsidered modifications were identified using 1% FDR as well. Identifications were transferred between samples using the ‘match between run’ option within a match window of 0.7 min and an alignment window of 20 min. Protein intensities for label-free quantification (LFQ) were calculated separately for each sample preparation method. The analysis was once repeated for the method comparison experiments allowing semi-specific tryptic digestion at the peptide N termini. Analysis of the MaxQuant results was done in Perseus (Version 1.5.0.31 and 1.6.1.1) (19Tyanova S. Cox J. Perseus: a bioinformatics platform for integrative analysis of proteomics data in cancer research.Methods Mol. Biol. 2018; 1711: 133-148Crossref PubMed Scopus (240) Google Scholar). At first, reverse hits, contaminants and proteins only identified by a modification site were removed. Afterward protein related parameters (identified proteins, quantified proteins, Pearson correlation coefficients, coefficients of variation), peptide related parameters (identified peptides, quantified peptides, missed cleavage sites, ragged-N peptides, modified peptides (C, oxM)) and dependent peptide related parameters (distribution of sample preparation-related peptide modifications) were extracted from the respective .txt files. For comparison of the different sample preparation protocols, the number of ragged-N peptides, modified peptides (C, oxM) and dependent peptides were normalized to the number of unmodified tryptic peptide identifications of the same run. Only peptides and proteins without missing values were considered for quantification. Significant protein expression differences between mouse lung samples were identified using FDR-adjusted p values from an ANOVA test with a permutation-based FDR of 0.01 and 250 randomizations. Significant differences between sample preparation methods were further analyzed by calculating the Tukey's honestly significant difference (THSD) using an FDR of 0.01. THSD were further used for gene ontology (GO) enrichment analysis using a Benjamini-Hochberg FDR threshold of 0.01. Quantification of bacterial species was based on summed iBAQ-intensitie