Analysis of protein mixtures from whole-cell extracts by single-run nanolc-ms/ms using ultralong gradients

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1 Analysis of protein mixtures from whole-cell extracts by single-run nanolc-ms/ms using ultralong gradients Thomas Köcher 1, Peter Pichler 2, Remco Swart 3 & Karl Mechtler 1,4 1 Research Institute of Molecular Pathology, Vienna, Austria. 2 Christian Doppler Laboratory for Proteome Analysis, University of Vienna, Vienna, Austria. 3 Dionex Corporation, Amsterdam, The Netherlands. 4 Institute of Molecular Biotechnology (IMBA), Vienna, Austria. Correspondence should be addressed to T.K. (thomas.koecher@imp.ac.at) or K.M. (karl.mechtler@imp.ac.at). Published online 12 April 212; doi:1.138/nprot The majority of proteome-wide studies rely on the high separation power of two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS), often combined with protein prefractionation. Alternative approaches would be advantageous in order to reduce the analysis time and the amount of sample required. On the basis of the recent advances in chromatographic and mass spectrometric instrumentation, thousands of proteins can be identified in a single-run LC-MS/MS experiment using ultralong gradients. Consequently, the analysis of simple proteomes or clinical samples in adequate depth becomes possible by performing single-run LC-MS/MS experiments. Here we present a generally applicable protocol for protein analysis from unseparated whole-cell extracts and discuss its potential and limitations. Demonstrating the practical applicability of the method, we identified 2,761 proteins from a HeLa cell lysate, requiring around 1 h of nanolc-ms/ms measurement time. INTRODUCTION Mass spectrometry (MS) has become a crucial tool in almost all areas of biological research, most notably by enabling the large-scale identification, characterization and quantification of proteins 1,2. In addition, major breakthroughs such as the complete characterization of the yeast interactome 3 led to the establishment of proteomics as an independent research field. The strategic orientation of biological research toward systems biology drives the development of advances in high-throughput technologies 4. The underlying hope for improved drug development and medical therapies is a strong driving force for massive investments in systems biology, including MS-based proteomics 5. Recent years have seen a quantum leap in functional proteomics, as demonstrated by the proteome-wide analysis of protein complexes 6 or chemical proteomics studies 7, in which small molecule target protein interaction networks are deciphered. In expression-based proteomics, changes in protein expression are analyzed. The high complexity of complete proteomes and the dynamic range of protein expression make these studies extremely challenging. The state-of-the-art analytical strategy in expressionbased proteomics relies on the analysis of peptides generated by in-solution digested proteins via online 2D-LC-MS/MS. This peptidecentric ( bottom-up ) approach is called shotgun proteomics and has predominantly replaced the traditional protein identification and quantification by 2D gel electrophoresis followed by in-gel digestion 8,9. Thousands of proteins can be identified by 2D LC-MS/MS from in-solution digested protein mixtures, but the very complex protein mixtures present in biological samples might require extensive protein and peptide fractionations 1. The drawbacks of the method are that large quantities of starting material are required and it is technically demanding to operate. Also the reproducibility over both dimensions is hard to achieve; therefore, the applicability of 2D LC-MS/MS to the label-free quantitation of clinical samples is debatable. Alternative routes to 2D LC-MS/MS, increasing the speed and throughput of the analysis, would be highly desirable and might be feasible owing to recent technological and methodological advances such as the introduction of extremely fast, sensitive and accurate mass spectrometers, complemented by advances in LC 11,12. MS-based strategies can be used to increase the analytical capacity of the mass spectrometer such as performing iterative LC-MS/MS runs and excluding previously identified peptides from the analysis or gas-phase fractionation 13. Although improved mass spectrometers have had an obvious effect on the depth of analysis, advances in LC instrumentation have been less adopted. The combination of state-of-the-art mass spectrometers with modern LC systems operated with ultralong gradient times and high peak capacities has, however, proven to be an extremely powerful technology 14, capable of identifying thousands of proteins in a single-run LC-MS/MS experiment 11,12,14. Twodimensional LC-MS/MS continues to be the method of choice for analysis of proteomes of higher organisms and might also be beneficial for protein quantification methods such as itraq (isobaric tags for relative and absolute quantitation) by reducing the distortion of reporter ion ratios caused by co-isolation of co-eluting peptides 15. Despite its limitations, we believe that single-run LC-MS/MS might be suited for the comprehensive analysis of simple proteomes such as yeast in the near future. The much shorter sample preparation, measurement and data analysis and favorable limits of detection make it an attractive option for biological applications requiring the analysis of less than a few thousand proteins. Experimental design The single-run LC-MS/MS method presented here is a temporal expansion of regular experiments. The workflow can be performed on any nanolc system (with pumps delivering at least 8 bar of pressure) coupled to any fast-scanning mass spectrometer. UV detection allows for monitoring the gradient formation, sample injection and evaluation of the separation of peptide standards. In the absence of a UV detector, MS-based detection can be used instead for quality control (QC) 16. LC-MS/MS-based protein identification can be divided into four main parts: (i) sample 882 VOL.7 NO nature protocols

2 preparation, including the proteolytic digest of the proteins; (ii) the online separation of the generated peptides by LC; (iii) the mass spectrometric analysis; and (iv) the bioinformatic interpretation of the data sets. Sample preparation. Sample preparation comprises a series of biochemical steps starting with lysis of the biological material and removal of other biomolecules such as lipids. However, in the context of shotgun proteomics, the final step is almost always the in-solution or the filter-aided 17 digest of the protein mixture. For the validation and optimization of the presented protocol, we used a tryptic digest of an acetone precipitated protein mixture from a HeLa cell lysate 11,16. The protein mixture is first digested with LysC, followed by digestion with trypsin, thereby generating peptides with either lysine or arginine at their C terminus 18. The two-step digestion of the protein mixture in the presence of urea aims for complete digestion. Quality control sample 1. We recommend preparing and storing a large amount of the aforementioned HeLa peptide mixture. This sample can be used for QC by injecting a defined amount (such as 1 µg) of peptides and monitoring the number of peptides and proteins identified with a specific false discovery rate (FDR). Quality control sample 2. To test the HPLC setup, we also recommend making a simple protein digest, such as a sample only containing digested cytochrome c. LC-MS/MS. LC-MS/MS experiments are performed by joining the LC system with a mass spectrometer. Consequently, for optimal results both instruments have to operate under perfect conditions, and the methods used on both instruments have to be adjusted to each other. Joining is achieved by ionization of the peptides with electrospray ionization (ESI) 19. The flow rate of the electrosprayed solvent is closely related to the ionization efficiency, with flow rates in the order of 1 nl min 1 yielding the highest mass spectrometric sensitivity 2,21. The optimal flow rate might be much higher; therefore, a compromise of a flow rate in the range of 2 to 3 nl min 1 is typically used. ESI emitters used for these flow rates have inner dimensions in the range of a few micrometers. Dedicated LC systems with all parts adapted to the low flow rates are commercially available (nanolc systems). High demands are imposed on nanolcs because even small dead volumes result in dramatic losses in resolution and, consequently, in the performance of the complete analysis. As a consequence, all nanolcs require dead volume free connections and all components such as capillaries, ferrules, sleeves, nuts and the columns must be of the proper format. The use of a configuration with an UV detector allows the evaluation of the performance of the LC system independently of the mass spectrometer. The UV detector is not essential for QC, but the immediate advantage of this configuration is that losses in performance can be located to either the mass spectrometer or the nanolc. Also, the programs used for interpretation of LC data allow the straightforward calculation of peak capacities and retention times. There are two fundamentally different possibilities for running the nanolc system, requiring a strategic decision. The first possibility is to directly load the peptide mixture from the autosampler to the analytical column in direct injection mode. The appeal of this configuration lies in its simplicity and ruggedness, but several drawbacks are associated with this approach. The low flow rate confined by the backpressure of the analytical column requires long loading times and maximum sample volumes of a few µl and does not allow for extensive washing. Consequently, this configuration requires off-line precleaning and preconcentration of the sample, facilitated by microcolumns 22. The organic solvent used for elution from these columns has to be removed before sample loading, which might result in sample losses. Precleaning of the sample allows directly combining the LC-column with the ESI emitter in a single capillary, strongly reducing postcolumn losses in chromatographic resolution. The second possibility is to use another configuration (Fig. 1), in which two different columns are used and two flow circuits are operated at different flow rates. The flow path can be changed by switching a valve. One column, the so-called trapping column, is used for concentration and cleaning of the sample. It can be operated at a much higher flow rates than the analytical column. In the sample loading step, the valve is switched in a way that the peptide mixture is transported from the sample loop to the trap column followed by washing (Fig. 1). The flow rate for loading and washing can be in the range of 5 to 2 µl min 1. At this point, the postcolumn flow is directed to the waste. When starting separation, the trapping column is switched in-line with the analytical column (Fig. 1). A gradient of increasing organic solvent is then applied for eluting peptides at a flow rate of 275 nl min 1. Trap columns allow the use of different solvents for loading and elution. It is recommended to use solvents containing trifluoroacetic acid (TFA) for loading, but not for eluting of the sample because of TSA s capacity for interference with ESI. The high ion-pairing strength of TFA helps minimize sample losses during loading phase. For the elution of peptides, formic acid or acetic acid is used. After the setup of the system, each flow path has to be flushed to ensure that there is no air in the system and to equilibrate the chromatographic material. The pressure sensors of the nanolc record the backpressure of the system. The resultant pressure curves can be used for evaluation of the system, such as the shape Load NanoLC pump Loading pump Elute NanoLC pump Loading pump Autosampler Autosampler Nano separation column Trap Nano separation column Trap Detector (UV/MS) Detector (UV/MS) Figure 1 The flow paths of the nanolc system. Flow paths are shown during sample loading and washing (top) and after switching for peptide elution (bottom). nature protocols VOL.7 NO

3 Figure 2 The UV chromatogram of a QC sample is shown together with the pressure curve of the nanolc separation. Key areas such as the elution window or the injection peak are assigned. The blue trace represents the pressure curve. of the injection profile (Fig. 2). Aberrant shapes point to incorrect fluidic connections within the nanolc system. Switching the valve from loading to elution (Fig. 1) should result in an increase of pressure (Fig. 2), which is caused by combining both columns. The pressure increase is preceded by a pressure decrease resulting from the compression of the additional volume. At the start of the gradient, the backpressure slowly increases to the maximum value, then slowly decreases, as determined by the different viscosity of the changing solvent composition. During the wash performed with a high percentage of organic solvent, the pressure decreases considerably and should return to the original value after the setup is switched back to the loading position. Simple peptide mixtures (e.g., quality control sample 2) should be used for QC of the system, such as a tryptic digest of cytochrome c, injecting UV-detectable quantities of 1 pmol (Fig. 2). We recommend evaluating at least four parameters: the pressure curves of the complete cycle, the peak heights, the chromatographic resolution and the retention times of the standard peptides. In the UV trace of the chromatographic separation of this QC sample, peak heights and chromatographic resolution can be directly assessed. The peak areas of the peptides can be used for analysis of their total recovery, indicating eventual performance issues of the autosampler. If the system performs properly, injecting a series of increasing amounts of a sample should lead to proportionally increasing peak areas. The gradient precision can be evaluated by performing repetitive LC experiments and analyzing the retention behavior of the peptides. Overlaying the UV traces of different runs provides the data for gradient precision, pointing toward eventual problems such as an unstable flow rate or inter-run composition of the gradient. Retention times of standard peptides should not differ by more than 1 s.d. from their mean values. If TFA is used as a loading solvent in a preconcentration setup, switching the analytical column in-line generates an UV signal because of its different UV absorbance. Prominent causes of LC failure are improperly assembled fluidic connections (especially when located postcolumn), damaged capillaries or other sources Absorption (mau) Dwell volume Inject peak Elution window Gradient delay Retention time / (min) Wash peak Equilibration Gradient at UV detector Programmed gradient of dead volumes. These can be detected by a sharp decrease and slow increase of the UV signal corresponding to TFA 23. Between LC runs, blank injections allow the monitoring of potential carryover of peptides between experiments. In many cases, we have found that issues with carryover can be solved by adding trifluoroethanol (TFE) to mobile phase B and using it to flush the injection system and the columns 24. Usually, MS equipment is set up by a service engineer from the manufacturer of the instrument by using off-line ESI with high flow rates and high voltage applied. In this mode the instrument is calibrated and other instrument-specific parameters are optimized. After fulfilling the respective specifications, the instrument should also be tuned in static nano-esi mode with a standard peptide at concentrations of 1 pmol µl 1. The MS/MS parameters for ion activation, such as optimal collision energies for collison-induced dissociation (CID), higher-energy collisional dissociation (HCD) 25 and electron transfer dissociation 26 should be optimized. After connecting the nanolc to the mass spectrometer via a nano-esi source, the background signals of the LC solvent should be recorded and relatively constant for several minutes. The spray should be stable and without the formation of droplets at the nano-esi tip. QC for the complete LC-MS/MS setup is finalized by evaluating its sensitivity. It is important to use correct quantities of the QC sample, adapted to the performance of the instrument. In the context of the described protocol we use 1 fmol of tryptic peptides generated from BSA, demanding 3% sequence coverage of BSA. After evaluating the sensitivity of the instrument, we apply another QC sample, this time 1 µg of tryptic peptides from a HeLa cell lysate, and evaluate the number of peptides and proteins Column pressure / (bar) a Peptides (no.) 2, 18, 16, 14, 12, 1, 8, 6, R 2 = , Gradient time / (h) b Proteins (no.) 3, 2,8 2,6 2,4 2,2 2, 1,8 1,6 1,4 1,2 1, R 2 = Gradient time / (h) c Peptides (no.) 2, 18, 16, 14, 12, 1, 8, 6, R 2 = , Peak capacity Figure 3 Triplicates of a CID-based LC-MS/MS analysis of 1-µg HeLa lysate using varying gradient times. Error bars represent 1 s.d. (a) Number of peptides identified plotted against the gradient time, following a logarithmic relation (R 2 =.99). (b) The number of proteins plotted against the gradient time. The relation follows a logarithmic relation (R 2 =.99). (c) The ratios between the experimentally obtained peak capacities and the number of identified peptides follow a linear relation (R 2 =.96). 884 VOL.7 NO nature protocols

4 Figure 4 LC-MS/MS analysis of varying amounts of HeLa lysate using HCD for fragmentation. Experiments were performed in triplicates and the gradient time was increased in steps from.5 to 1 h. Error bars represent 1 s.d. (a) The number of peptides identified from 1 µg (blue),.1 µg (red) and.1 µg (green) of HeLa lysate in.5, 1, 5 and 1 h (Fig. 3). (b) The number of proteins identified from 1 µg (blue),.1 µg (red) and.1 µg (green) of HeLa lysate in.5, 1, 5 and 1 h. a Peptides (no.) 16, 14, 12, 1, 8, 6, 4, 2, Gradient time / (h) b Proteins (no.) protocol 1 µg.1 µg.1 µg 1 µg.1 µg.1 µg 3, 2,5 2, 1,5 1, Gradient time / (h) identified with a specific MS/MS method and LC gradient (Fig. 3). It should be noted that optimization of the gradient length and the MS/MS settings are interdependent. Longer gradients lead to broader peak widths, which may require longer ion injection times to collect a desired target number of precursor ions. By using the described protocol and gradient times between 1 and 1 h, we observed a linear relationship between gradient time and peak widths. The mean peak widths at 4σ of an assumed Gaussian peak (peak width at 13.5% of the maximum peak height) were.23 min using a 1-h gradient, and they increased to.86 min for the 1-h gradient. In addition, the amounts and complexity of the sample should be considered. If samples from gel bands are analyzed, complexity is not an issue and steep gradients will perform best, providing optimal sensitivity. The decreased sensitivity of LC-MS/MS with long gradients is clearly visible in the HCD-based analysis of a.1-µg HeLa digest. Extending the gradient time from 1 to 1 h dramatically decreased the number of proteins identified (Fig. 4). MATERIALS REAGENTS Acetonitrile, HPLC grade (Merck)! CAUTION It is flammable and toxic. Wear appropriate gloves and safety goggles and use in a fume hood. Formic acid, 98 1% (Suprapur, Merck)! CAUTION It is corrosive. Wear appropriate gloves and safety goggles and use it in a fume hood. Milli-Q deionized water (Millipore) TFA (Pierce)! CAUTION It is corrosive. Wear appropriate gloves and safety goggles and use in a fume hood. Trifluoroethanol (TFE, Sigma-Aldrich)! CAUTION It is flammable and toxic. Wear appropriate gloves and safety goggles and use in a fume hood. EQUIPMENT NanoLC system (UltiMate 3 RSLCnano LC system (Dionex, part of Thermo Fisher Scientific) Modules comprising the NanoLC system NCS-35RS binary nano flow pump with ternary loading pump and column compartment WPS-3TPLRS Thermostatted pulled loop well-plate sampler VWD-34RS four-channel variable wavelength detector with a UZ-view flow cell nano, 3-nl volume, 1-mm path length SRD-34 integrated solvent and degasser rack, four channels Precolumn (trapping column): PepMap C18; 2 cm 1 µm 5 µm, 1 Å (Dionex, part of Thermo Fisher Scientific) Analytical column: Acclaim PepMap RSLC C18; 5 cm 75 µm 2 µm, 1 Å (Dionex, part of Thermo Fisher Scientific) Software for controlling and interpreting nanolc: Chromeleon Chromatography Data System (v.6. 8.; Dionex, part of Thermo Fisher Scientific) Finally, the mass spectrometric raw data have to be automatically interpreted to identify the peptides and the proteins present in the sample. Identification is based on both the exact peptide mass and the tandem mass spectrum generated 27. For this purpose, we have used Mascot software, but a number of other search engines such as Sequest, X!Tandem or Phenyx exist 28. A number of quality parameters can be used to minimize the number of false-positive identifications, while maximizing the outcome of the search. Approaches that translate the output of the search engine in either peptide and protein probabilities or estimated FDR have been developed 28. On the basis of the target-decoy approach 29, we restricted the FDR of both peptides and proteins to 1%. It should be noted that at present commonly accepted rules for protein identification do not exist; however, scientific journals have adopted their own standards 3. Although many practitioners use an FDR-based filtering, other approaches such as the so-called two-peptide requirement are still used 31. Mass spectrometry Nanoelectrospray source (Proxeon) Nanoelectrospray emitters: PicoTip tubing outer diameter (o.d.)/inner diameter (i.d.) 36 µm/2 µm, tip i.d. 1 µm (New Objective, no. FS D-2) LTQ-Orbitrap Velos (Thermo Fisher Scientific) Software for controlling MS: XCalibur (v.2.1..; Thermo Fisher Scientific) and Tune (v.2.6..; Thermo Fisher Scientific) Software for interpreting MS/MS data sets: Proteome Discoverer (v.1.3..; Thermo Fisher Scientific) Software for searching MS/MS data sets: Mascot (v ; Matrix Science) REAGENT SETUP Quality control sample 1 Prepare and store a large amount of a complex standard sample, such as peptides generated from HeLa cells. The peptide mixture was generated as described previously 11, and a procedure can be found in a protocol available online 32 ( protex.212.1). Aliquots can be stored frozen at 8 C for a maximum of 2 years with a protein concentration of 1 mg ml 1. Quality control sample 2 A simple protein digest such as peptides proteolytically generated from 1 pmol of cytochrome c and 1 fmol of BSA. A stock solution can be stored at 8 C for a maximum of 6 months. Sample to analyze In principle, all protein mixtures can be analyzed after proteolytic digestion; however, the presence of detergent in the sample buffer, especially sodium dodecyl sulfate (SDS), should be avoided. With the nature protocols VOL.7 NO

5 described protocol and column dimensions, the amounts of injected peptide mixtures should not be >5 µg. NanoLC pump mobile phase A Mobile phase A is freshly prepared acetonitrile (2% (vol/vol)),.1% (vol/vol) formic acid in Milli-Q deionized water. NanoLC pump mobile phase B Mobile phase B is freshly prepared acetonitrile (8% (vol/vol)),.8% (vol/vol) formic acid and 1% (vol/vol) TFE in Milli-Q deionized water. Loading pump mobile phase Loading pump mobile phase consists of freshly prepared TFA (.1% (vol/vol)) in Milli-Q deionized water. EQUIPMENT SETUP NanoLC setup The splitless UltiMate 3 RSLCnano LC system consists of two different pumps. The loading pump has a pressure limit of 5 bar and delivers the loading solvent. The HPG Nano Pump delivers the gradient and has a pressure limit of 8 bar. Further, the system comprises a thermostatted column compartment containing up to two high-pressure 1-port switching valves, a UV detector with a 3-nl flow cell volume, an autosampler equipped with a sample loop available in sizes between 2 and 125 µl, an injection needle in sizes between 2.5 and 15 µl and a 25-µl syringe. For sample injection from the autosampler, a loading solvent of.1% (vol/vol) TFA in water is used. A scheme of the fluidic connections is shown in Figure 1. All connections from the analytical column to the mass spectrometer inlet have to be of a 2-µm i.d. format using zero-dead-volume micro-tight connectors. To connect the UV cell outlet with the nano-esi source, use fused silica (2 µm i.d.) and keep the connection as short as possible. Before using the nanolc for sample analysis, switch on the system and equilibrate the analytical column with mobile phase at a flow rate of 5 nl min 1. The UV lamp is also switched on and, if required, the signal intensity is set to zero after stabilization. Increase the flow rate to 275 nl min 1 and run the system until the pressure and UV baseline are stable. The sample is first loaded from the sample loop onto a trapping column (Acclaim PepMap C18; 2 cm 1 µm 5 µm, 1 Å) using the loading solvent and a loading flow rate of 5 µl min 1 for 1 min. Sample loading volumes depend on the loop size and should be calculated. The trapping column is then switched in-line with the separation column (Fig. 1) and the peptides are eluted with increasing organic solvent at a flow rate of 275 nl min 1. The peptide separations are carried out at 3 C using a 75-µm ID 5 cm C18 column (Acclaim PepMap RSLC C18, 5 cm 75 µm 2 µm, 1 Å) mounted in the thermostatted column compartment. A gradient of increasing organic solvent is used, as described in Table 1. Gradient programs for other gradient times are calculated by simple extension of this linear gradient. This is followed by a second short gradient, increasing the percentage of phase B to 9% in 5 min. Mass spectrometer setup The mass spectrometer LTQ-Orbitrap Velos was operated in positive ionization mode. Single MS survey scans were performed in the Orbitrap, recording a mass window between 35 and 2, m/z using a maximal ion injection time of 4 ms. The resolution was set to 6, and the automatic gain control was set to 1,, ions. The lock mass option was enabled allowing the internal recalibration of spectra recorded in the Orbitrap by polydimethylcyclosiloxane background ions (protonated (Si(CH 3 ) 2 O)) 6 ; m/z ) 33. The experiments were done in data-dependent acquisition mode with alternating MS and MS/MS experiments. The minimum MS signal for triggering MS/MS was set to 5 ions, with the most prominent ion signal selected for MS/MS using an isolation window of 1.9 Da. The m/z values of signals already selected for MS/MS were put on an exclusion list for 18 s using an exclusion window size of ±5 p.p.m. In all cases, one micro-scan was recorded. CID was done with a target value of 5, ions in the linear ion trap, a maximal ion injection time of 2 ms, normalized collision energy of 35%, a Q-value of.25 and an activation time of 1 ms. A maximum of 2 MS/MS experiments were triggered per MS scan. HCD was done with a target value of 5, ions (and recorded in the Orbitrap), a maximal ion injection time of 25 ms and a normalized collision energy of 35%. A maximum of 1 MS/MS experiments were triggered per MS scan. Bioinformatic analysis We searched raw files with Proteome Discoverer (v ) using the search engine Mascot For HeLa samples, Table 1 One- and five-hour gradient elution program for nanolc separation. Time (1-h gradient) Time (5-h gradient) Gradient composition Switch valve position min min 2% B 1_2 1 min 1 min 2% B 1_1 65 min 35 min 4% B 7 min 31 min 9% B 75 min 315 min 9% B 77 min 317 min 2% B 8 min 32 min 2% B 1_2 15 min 345 min 2% B The flow rate, gradient composition and 1-port switching valve position programming are listed (see Fig. 1). The flow rate is constant at 275 nl min 1. the database included all human sequences of Swiss-Prot release 211_2, supplemented with a set of common contaminant proteins as well as a decoy version of all sequences. The SequenceReverser.exe tool within MaxQuant was used to create a concatenated forward and reversed database containing 41,462 sequences. Precursor mass tolerance was 5 p.p.m., and fragment ion tolerance was.5 Da for CID recorded in the ion trap and 5 mda for the HCD spectra recorded in the Orbitrap. A maximum of two missed cleavages was permitted using trypsin as the enzyme. In addition to b-ions and y-ions, water and ammonia losses from both ion series were scored. Carbamidomethylation of cysteine residues was set as a fixed modification, and oxidation of methionine was set as a variable modification. Further variable modifications such as carbamylation of peptide N termini and lysine residues, or deamidation of glutamine and asparagines may be included in the search. However, expansion of the number of variable modifications leads to a combinatorial enlargement of search space. This may cause an increase in the number of false-positive identifications, requiring more stringent filter criteria to keep the FDR constant. Thus, the net result might be an overall decrease in the number of identified peptides when the fraction of modified peptides is low. We therefore do not recommend searching for many different types of variable modifications. Initial import filters for peptide-spectrum matches (PSMs) were a rank 1 peptide and a minimum length of 7 amino acids. The initial confidence parameter was defined by a minimum Mascot score achieving an FDR of 1% for PSMs, calculated manually as the number of spectra that map to a decoy protein divided by the number of spectra mapping only to actual protein sequences. Proteins were grouped according to the maximum parsimony principle using the protein grouping algorithm implemented in Discoverer , which ensures that each spectrum may only contribute evidence to the existence of a single protein group and that the smallest set of proteins is reported that explains all the spectra evidence. Protein grouping was performed on the subset of PSMs with a Mascot score, which was sufficient to keep the FDR at 1% for the identified protein groups. Other search engines and software 28 can be used for data processing, such as tools within the Trans- Proteomic Pipeline 34. These tools include Peptide Prophet 35, for estimating the confidence of PSMs; Protein Prophet 36, for inferring the protein result; and Max Quant 37, a program initially developed for protein identification. For QC of the LC data sets such as the UV chromatogram the LC software package, we used Chromeleon. 886 VOL.7 NO nature protocols

6 PROCEDURE System setup of the nanolc 1 If the system was not in use, purge the pumps and flush the complete system with freshly prepared and degassed mobile phases to ensure that there is no air in the system. 2 Attach a new precolumn (trapping column) to the nanolc system by connecting the respective tubing (Fig. 1) and pass 1 µl of solvent A at a flow rate of 5 µl min 1 through the system and into the waste. 3 Attach a new nanolc column to the system by connecting the respective tubing (Fig. 1) and pass a 6-µl volume of solvent B followed by 6 µl of solvent A through the system. Start with lower flow rates (5 nl min 1 ) to keep the system well below the pressure limit. Use a final flow rate of 275 nl min 1. 4 Perform two replicates of the injections by switching the trapping column in-line with the analytical column to monitor the backpressure of the system and the UV signal recorded. The injection peak (Fig. 1) should be reproducible (if a loading solvent with a composition different from mobile phase A is used, a UV signal caused by switching the trapping column in-line with the analytical column is visible). The UV absorbance should reach baseline levels after the injection peak. 5 Equilibrate the nanolc system with 98% solvent A using a flow rate of 275 nl min 1 on the analytical column. Wait to reach a stable backpressure, which should be well within the specifications of the system (note that the pressure will rise after loading the column, when increasing the acetonitrile concentration and during the lifetime of the column). 6 Program a method using a short gradient with a 3-min peptide separation, and which contains an appropriate washing procedure for the syringe and other components of the autosampler (see EQUIPMENT SETUP). 7 Inject samples via the autosampler using a loading flow rate of 5 µl min 1 (see EQUIPMENT SETUP). Perform several replicates of LC analyses using short gradient times of 1 pmol of a protein digest (note that new columns have to be saturated with peptides because of free active sites of the stationary phase). QC of the nanolc system using UV detection (optional) 8 Before the analysis of biological relevant samples, perform a QC check of the system with a well-characterized sample such as a tryptic digest of cytochrome c. Monitor the retention time of the peptides and their precision. Retention times should be stable and the relative standard deviation should be <.2% or.1 min s.d. 9 Perform blank injections and monitor the background UV signal of the LC analysis. Setup and QC of the complete LC-MS/MS system 1 Connect the nanolc system to the mass spectrometer via a nano-esi ion source (see EQUIPMENT SETUP). 11 Monitor the background single-ms signal of the mass spectrometer, which should have an instrument-specific intensity. 12 Program a MS/MS method for the mass spectrometer, triggering acquisition by the LC software and aligning the start of the nanolc gradient with the start of MS/MS acquisition. MS/MS should be performed in data-dependent acquisition mode (see EQUIPMENT SETUP). 13 Perform sensitivity tests of the complete nanolc-ms system by injecting 1 fmol of BSA digest and running a 3-min LC separation. Interpret the raw data from two consecutive runs (see EQUIPMENT SETUP). Monitor the sequence coverage and the number of peptides identified. These numbers are characteristic for a specific nanolc-ms/ms setup and should be relatively constant. nature protocols VOL.7 NO

7 Optimize gradient length and MS/MS program with a complex QC sample 14 Program several methods for the nanolc using long gradient times that define the optimal procedures for a given sample complexity and quantity (see EQUIPMENT SETUP). 15 Optimize the MS/MS parameters for maximum numbers of identified proteins. 16 Run LC-MS/MS experiments for the different gradient times in replicates. The QC sample should mimic the properties of the real biological sample. 17 Analyze the results (see EQUIPMENT SETUP). Troubleshooting advice can be found in Table 2. Table 2 Troubleshooting table. Step Problem Possible reason Solution 2 Pressure fluctuations are observed for the loading pump 3 Pressure fluctuations are observed for the nanolc pump 4 Slow increase of UV absorbance to reach baseline Slow return of column pressure after switching trapping column in-line Air in the loading pump Air in the nanolc pump Dead volume in the trapping column Loose connection of the trapping column Purge the loading pump Purge the nanolc pump Check all connections; check the column pressure traces (trap and nano column) Reconnect the trapping column; check the column pressure traces (trap and nano column) 5 Column pressure is not built up Leak in the fluidic setup Check the fluidic connections Maximum system pressure is reached Column or emitter blockage Check the column and emitter and replace if necessary 7 Large pressure drop loading pump Air is present in the sample loop Check the injection program and sample vial; flush the autosampler NanoLC pump pressure increase Column or emitter blockage Check the column and/or spray emitter and replace if necessary 8 No peptide retention Organic solvent is present in the sample loop or sample liquid Check the injection program and sample solvent Trapping column is not equilibrated Check the program and increase the trapping column equilibration time Nonreproducible retention of peptides Column is not equilibrated Increase the column equilibration time and check the column pressure profile Temperature fluctuations Use the column oven Broad peaks Postcolumn dead volume Check the column-to-emitter connection Only late eluting peptides are observed Breakthrough of hydrophilic peptides on the trapping column Increase the ion-pairing agent concentration Increase in trapping column pressure All peptides elute late with acceptable chromatography (chromatogram shifted) All peptides elute late with unacceptable chromatography The sample contains particulate material or undigested protein Dead volume before the (trap) column (additional gradient delay time) Dead volume after the nanolc column Improve sample preparation (filtration/ digestion) Check all fluidic connections of the trap column and upstream Check all fluidic connections postcolumn 9 Undefined peaks are detected in the gradient of the blank injection Contaminated solvent Refresh the LC/MS-grade solvents Peptides appear in the blank gradient Carryover Use the wash solvent for the trapping column and/or autosampler needle 888 VOL.7 NO nature protocols

8 ANTICIPATED RESULTS Here we presented a nanolc-ms/ms protocol and QC measures for optimizing the identification of proteins in a single LC-MS/MS experiment. We used a HeLa cell lysate for evaluating the limits and the potential of the method because it provides us with a very high number of detectable peptides, estimated to be in the range of 1, (ref. 38). HeLa is commonly used for evaluation of protein identification routines, facilitating direct comparisons. The method can be readily used for other peptide mixtures and is easily adaptable to any state-of-the-art LC-MS/MS equipment. Proteins are identified indirectly via sequencing of online separated peptides by MS/MS. Logically, the limits of the method are largely defined by the acquisition speed of the mass spectrometer, its sensitivity and the total time of the LC separation. However, these parameters are interconnected because the total time of the LC separation will lead to an increase of the peak widths of the eluting peptides, negatively affecting the signal intensity. Investigating the effects of increasing gradient times using CID on an LTQ Orbitrap Velos instrument, we observed a logarithmic increase of peptide identifications with time (Fig. 3a) 11. By using 1 µg of tryptic HeLa peptide mixture, we identified an average number of 5,796 peptides with a 1-h gradient compared with a maximum of 18,13 peptides with a 1-h gradient. This corresponded to the identification of 1,236 and 2,761 proteins, respectively (Fig. 3b). Interestingly, the relation between the number of identified peptides and the used gradient time is not linear, whereas the relation between the number of identified PSMs and the gradient time is almost linear (data not shown, R 2 =.94), with an increasing number of PSMs identifying redundant peptide sequences. While investigating the interplay between identified peptides and parameters of the chromatographic separation, we observed a linear relation between the number of identified peptides and the peak capacity (Fig. 3c). This observation stresses the importance of the required excellent peptide separation capabilities in LC-MS/MS using long gradient times. By analyzing the same peptide mixture with HCD, we were able to evaluate the system regarding its sensitivity. As mentioned above, long gradients result in increasing peak widths, generating less ions for a given ion injection time. We used HCD for this purpose because although HCD generates high-quality spectra with high mass accuracy, it requires more ions for successful peptide identifications. We analyzed and compared the results for.1,.1 and 1 µg of injected peptide mixture using a.5-, 1-, 5- and 1-h gradient (Fig. 4). Comparing the results obtained with HCD and CID for the 1 µg sample, we identified slightly fewer proteins and peptides using HCD, on average around 8% at the protein level. This result confirms initial observations for this mass spectrometer when using 2 µg of a HeLa digest 39. Naturally, the relative protein identification efficiency of HCD also depends on the sample amounts and the settings used for MS/MS acquisitions. Apart from again observing a logarithm time-dependence for peptide and protein identifications with increasing gradient times with a.1- and.1-µg sample, we observed that for.1 µg the 5-h gradient already led to the identification of fewer proteins than a 1-h gradient (Fig. 4). Undoubtedly, this effect is caused by increasing peak widths for longer gradient times, thereby resulting in signal intensities that are not sufficient for successful protein identification. This result also underscores the importance of evaluating the complete LC-MS/MS method with a QC sample reflecting the amounts and the composition of the actual sample to be analyzed. Different gradient times should be evaluated because the most efficient procedure is not always the method with the longest gradient and analysis time. Rather, the amount and the complexity of the sample influence the optimal choice of the gradient time and the fragmentation method. The negative effect of increasing peak widths and lower peak intensities for longer gradient times is also evident in CID-based methods, although CID requires fewer ions for a tandem MS spectrum of acceptable quality. In the same context, it is also interesting to note that the difference between the.1-µg and 1-µg sample is substantially less pronounced for the.5- and 1-h gradient. Here the results indicate that for short gradient times, the duty cycle is determined mainly by the time required to transfer ions and acquire the image current in the Orbitrap rather than by the ion injection time required to collect the specified number of ions. Given the information obtainable with relatively small investments of analysis time and amounts of protein sample, we anticipate that single run LC-MS/MS methods will be widely used for the initial characterization of proteomes. It might eventually also be possible to analyze complete proteomes of some organisms such as bacteria or even yeast, after further technological improvements in LC and MS instrumentation. Acknowledgments This work was funded by Boehringer Ingelheim, the Christian Doppler Research Association, the Austrian Proteomics Platform within the Austrian GenomeResearch program (GEN-AU), the Austrian Science Fund via the Special Research Program Chromosome Dynamics (SFB-F342) and the European Commission via the FP7 projects MeioSys and Prime XS. The technical support of the other members of the Mechtler group, especially of G. Krssakova, is gratefully acknowledged. AUTHOR CONTRIBUTIONS T.K. and K.M. designed the study. T.K. and P.P. performed experiments and analyzed the data. R.S. provided conceptual input and assisted in the experimental design. T.K. and K.M. supervised the project. All authors discussed the experimental results. T.K. wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at Reprints and permissions information is available online at com/reprints/index.html. 1. Gstaiger, M. & Aebersold, R. 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