Accurate, High-Throughput Protein Identification Using the Q TRAP LC/MS/MS System and Pro ID Software

Transcription

1 Proudly serving laboratories worldwide since 1979 CALL for Refurbished & Certified Lab Equipment ABI Q Trap Pro LC/MS/MS Accurate, High-Throughput Protein Identification Using the Q TRAP LC/MS/MS System and Pro ID Software Purpose This application note demonstrates automated protein identification from complex proteomic samples using the Q TRAP LC/MS/MS System and Pro ID software. Overview Researchers have traditionally performed protein identification by liquid chromatography mass spectrometry using three-dimensional (3-D) quadrupole ion traps or hybrid quadrupole time-of-flight mass spectrometers such as the API QSTAR Pulsar LC/MS/MS system. Instruments such as the QSTAR system offer premium performance, while 3-D quadrupole ion traps, because of their lower cost, have gained widespread acceptance. Conventional 3-D ion traps, however, have several limitations for proteomic applications. For example: Mass accuracy is poor, resulting in ambiguous database search results. Important low mass ions are not observed in typical MS/MS experiments. Longer cycle times are incurred when performing higher-resolution scans, with the increased resolution providing no increase in mass accuracy. True precursor ion and neutral loss scans, useful for protein characterization, are not available. The Q TRAP LC/MS/MS System is a unique system solution that provides enhanced performance to rapidly identify more peptides and proteins per sample. Based upon new linear hybrid technology patented by Applied Biosystems/MDS SCIEX, the Q TRAP system combines superior ion trap capabilities with the powerful scan modes of a triple quadrupole mass spectrometer. In this versatile instrument, typical ion trap capabilities of high sensitivity are present without sacrificing mass accuracy and resolution. In addition, all normal triple quadrupole features including true precursor ion and neutral loss scans are maintained. Coupled with new intuitive acquisition software and Pro ID software for automated processing, the result is more proteins identified with higher confidence in less time. Key Features Enhanced resolution and mass accuracy compared to conventional 3-dimensional ion trap and standard triple quadrupole mass spectrometers, which provides greater confidence in your database search results Superior full-scan sensitivity in MS and MS/MS modes permits analysis of important low-copy proteins Standard triple quadrupole-like MS/MS fragmentation with no low mass cutoff provides better peptide sequence coverage, improving database search results Unique scan functions that include MS3, neutral loss, precursor ion, and multiply charged scans provide more flexibility for protein identification and characterization Pro ID software with patented Interrogator search algorithm allows rapid, accurate protein identification, even from proteins with multiple modifications

2 Application-specific software provides ease-of-use Peptide Infusion Experiments A standard mixture of 26 synthetic peptides (ranging in size from 700 to 2,200 Da) was analyzed by nanoflow infusion (0.5 L/minute) with the Q TRAP system as well as a conventional 3-D ion trap under identical conditions. Full scan MS spectra were acquired on both instruments, where the peptide mixture was diluted at several concentrations as shown in Table 1. The identical sample solution, infusion pump, and tubing were used for both instruments. For the Q TRAP system, we acquired the MS data using the novel Enhanced Multiply Charged (EMC) Scan as the MS scan. The EMC scan is a new scan type that eliminates the majority of singly charged ions, which in turn, maximizes the signal-to-noise ratio for multiply charged ions. For tryptic peptides using electrospray ionization, the vast majority of the m/z ions will be multiply charged, so this type of scan results in extremely high sensitivity for peptides in a complex mixture. The table shows that both instruments identified a similar number of the 26 individual peptides at high concentrations. However, this dropped off dramatically as the concentration of the peptide mixture decreased. Clearly, the Q TRAP system outperformed the conventional 3-D ion trap for full-scan MS sensitivity. Figure 1 compares the spectra obtained for the peptide mixture at a concentration of 5 fmol/ L. The signal-to-noise ratio of the ions in the spectrum for the Q TRAP system is far superior to that obtained on the 3-D ion trap, and hence, many more of the peptide ions are observed. In total, the Q TRAP system identified 22 of the 26 peptides in the EMC spectrum, while the 3-D trap identified only three. Yeast Proteome LC/MS/MS Experiment We analyzed the proteome of wild-type yeast (Saccharomyces cerevisiae) with the Q TRAP LC/MS/MS system and processed the results with Pro ID software. Yeast was grown to mid-log phase (OD600 = 0.7) and yeast extracts were prepared using the liquid nitrogen (LN2) grinding method. The resultant protein samples were labeled with iodoacetamide and digested with trypsin. After digestion, we separated the samples into 25 fractions by ionexchange chromatography using a Vision workstation. Experimental Conditions Mass spectrometer: Q TRAP LC/MS/MS System Ion source: Nanospray interface Column: 75 m ID x 150 mm PepMap, C18, 3 m from LC Packings LC pumps, autosampler, flow rate, injection volume: 250 nl/min using LC Packings

3 Ultimate LC pump, injecting 20 L on column using LC Packings Famos autosampler. Mobile phases: A=0.1% formic acid; B=90% acetonitrile + 0.1% formic acid Gradient: 2 5% B in 5 minutes; 5 30% B in 65 minutes; 30 90% B in 5 minutes Vision Workstation: One-minute time points were collected into separate fractions using a 4 x 15 mm HS50 column; a 2 ml square well rack; a dual wavelength UV detector; and a 3 mm/4.5 L analytical flow cell Data acquisition: Enhanced MS survey scan (~1 second) followed by two dependent Enhanced Resolution scans (~1 second each) and two dependent Enhanced Product Ion scans (~2 seconds each) giving a total cycle time of about 6 seconds. IDA Criteria: 400 1,700 m/z scan range; consider charge states 2 and 3 only; dynamically exclude former target ions for 60 seconds; mmu mass tolerance; and ignore isotopes for all previously fragmented ions. Results and Discussion We loaded cation exchange fractions into the LC Packings autosampler and automatically injected them onto the Q TRAP system for data analysis. We programmed the instrument with the IDA Method Acquisition Wizard to automatically perform one Enhanced MS (EMS) survey scan, two Enhanced Resolution (ER) scans, and two Enhanced Product Ion (EPI) scans throughout the entire LC/MS/MS run. Enhanced scan modes use the trap capabilities of the instrument to improve sensitivity, resolution, and mass accuracy. Upon completion of data acquisition, the Pro ID software program automatically processes the data for protein identification. For each ER scan, Pro ID determines the charge state and accurate mass (calculated average mass accuracy from Figure 2 is 75 ppm) of the precursor, and thus the peptide molecular weight, and uses the corresponding MS/MS data from those precursors to find matches to peptides in a protein or DNA database. Pro ID processes all cycles in the data and creates a summary report consisting of a list of identified proteins and the peptides that were identified with their confidence values. In addition, all results are saved in a relational database to allow easy data retrieval and comprehensive queries. Depending upon the size of the database searched and the number of modifications included in the search, the processing of a typical one-hour LC/MS/MS data file by Pro ID takes 1 10 minutes. Because of its ease of cultivation, genetic manipulation, and short generation times, yeast is an ideal system for the study of biological processes relevant to higher eukaryotes. The complete genome of yeast is known, and research efforts continue to characterize its complete proteome. Table 2 categorizes the proteins identified in this study and the percentages of each identified from the cation exchange fractions combined. Proteins typically considered to be of high abundance as well as some lower abundance proteins are observed. Figure 2 displays a portion of the Pro ID Protein Summary from the analysis of one cation exchange fraction from the yeast protein mixture. Figure 2 also depicts the expansion of the proteins pyruvate kinase and enolase 2 to show the individual peptides that were identified. Pyruvate kinase was identified from ten different MS/MS spectra and enolase 2 was found from four spectra. The peptide sequences that correspond to the MS/MS spectra are listed in the expanded results as shown in Figure 2. The more peptides identified that match the same protein, the more confidence there is in the protein ID result. Using the right-click menu Show ID evidence lets you compare the MS/MS fragment data to its peptide sequence in a BioAnalyst software peptide sequencing window. Figure 3 shows that, out of 26 possible theoretical b and y ions, 21 were found in the MS/MS data for the peptide with sequence EPVSDWTDDVEAR from pyruvate kinase. In addition, many supporting fragment ions, such as y- NH3 and low-mass immonium ions typically not seen in a traditional 3-D ion trap, match the data all increasing the confidence that the correct protein has been identified.

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6 Yeast contains over 6,000 open reading frames (ORFs). In one cation exchange fraction, 120 proteins were identified with a confidence of 90 or higher using an MS tolerance of 0.5 and MS/MS tolerance of 0.3 Daltons for the database search. In comparison, a search of similar data acquired using a 3-D ion trap required larger MS (1.5 Da) and MS/MS (0.7 Da) tolerances due to the lower mass accuracy. For the yeast data, the fragment ion mass accuracy on the Q TRAP system was ±0.1 Da, while for the 3-D ion trap it was ±0.75 Da. The 3-D ion trap identified only 62 proteins with a confidence of 90 or higher only 51% as many as the higher mass accuracy data from the Q TRAP system. For the abundant protein HSP 70, the Q TRAP System identified 12 peptides that matched to this protein, while the 3-D ion trap found only 5. The Q TRAP system obtained better protein coverage, increasing confidence in the identification. Conclusions The Q TRAP LC/MS/MS System is a new linear ion trap hybrid mass spectrometer that outperforms 3-D ion traps and delivers high-performance triple quadrupole functionality. This powerful platform provides high sensitivity for protein identification for proteomics applications. Coupled with Pro ID and BioAnalyst software, the Q TRAP system lets you identify more proteins faster and with higher confidence. Furthermore, the unique scan functions of the Q TRAP system enable highly specific biomolecule identification and characterization. Now you can go straight to the answers.

7 Introduction to the Q Trap LC/MS/MS System The Q Trap LC/MS/MS system is a hybrid triple quadrupole linear ion trap (LIT) mass spectrometer. The Q3 region can be operated as either a standard quadrupole mass spectrometer or a linear ion trap mass spectrometer. The unique scan modes of both configurations can be linked to provide more and higher quality data than either instrument alone. For example, a precursor ion scan in Transmission mode can be used as a survey scan in order to target specific ions to be used in an enhanced product ion scan (in LIT mode). Conversion between the two modes of operation is rapid, since it involves only the addition or removal of the resolving DC voltages. The Q Trap LC/MS/MS system retains all of the traditional triple quadrupole scan types such as: Q1 MS (Q1) Q1 Multiple Ion (Q1 MI) Q3 MS (Q3) Q3 Multiple Ion (Q3 MI) Multiple Reaction Monitoring (MRM) Precursor Ion (Prec) (This is not possible with a conventional ion trap.) Product Ion (MS2) Neutral Loss (NL) When Q3 operates as an LIT mass spectrometer, a number of new advantages and capabilities are available: High sensitivity product ion scanning Fast scanning (4000 amu per second) High resolution capabilities at reduced scan speeds MS/MS/MS capabilities Reduced space charge effects In LIT mode, a pulse of ions is introduced into the ion trap. The main RF fields trap the ions in the radial direction, while DC voltages applied to the lenses at both ends of Q3, trap the ions axially. The trapped ions are allowed to cool for several milliseconds, then the RF voltage is scanned in the presence of a low voltage auxiliary AC applied to the rods. The ions ejected axially toward the detector are counted. If you configure the mass spectrometer with Q1 operating as a standard quadrupole mass spectrometer and Q3 operated as an LIT mass spectrometer, you can achieve the following enhanced scan types: Enhanced MS (EMS) Enhanced Resolution (ER) Enhanced Product Ion (EPI) Enhanced Multi-Charge (EMC) Time Delayed Fragmentation (TDF) MS/MS/MS (MS3) In LIT mode, a pulse of ions passes through Q1 operated as a conventional quadrupole mass spectrometer to select the precursor ion of interest. The precursor ions are accelerated into the pressurized Q2 to promote fragmentation. The fragment and residual precursor ions are then trapped in the Q3 linear ion trap. The Q3 RF voltage is ramped and the ions ejected toward the detector are reported. For more information about these enhanced scans, see Q Trap LC/MS/MS Enhanced Modes of Operation on page 10.

8 Triple Quadrupole/Linear Ion Trap Mass Spectrometer The Q Trap LC/MS/MS system uses a TurboIonSpray, Heated Nebulizer, or Flow Nanospray ion source to produce ions from liquid samples. The term LC/MS/MS, applied to the triple quadrupole series, is a generic label for the combined analytical processes of liquid separation and subsequent mass spectrometric analysis. The instrument is configured to perform complex MS/MS and MS/MS/MS analysis. For less rigorous analytical requirements, it can perform single MS (LC/MS) scans. The Q Trap LC/MS/MS system allows all modes of MS/MS and MS/MS/MS operation for full characterization of biopharmaceutical compounds and the specificity needed for new drug development. For pharmaceutical and pharmacokinetic samples, MS/MS has the sensitivity and specificity required to analyze hundreds of samples per day without extensive sample preparation. For peptides and proteins, molecular weights can be determined with accuracies better than 0.01% at 200 kda. The major components of the Q Trap LC/MS/MS system are shown in the figure Q Trap LC/MS/MS system components with pump on page 7. Principles of MS In Single Quadrupole mode, the Q Trap LC/MS/MS system separates ions representative of the sample molecular components based on their m/z ratio. Ions of a unique m/z ratio can be separated by the single mass filter quadrupole and counted to provide mass spectra for the sample. The mass filter quadrupole consists of four cylindrical rods mounted in a ceramic collar surrounding the ion path. Fixing the ratio of RF to DC voltages applied to the quadrupole rods determines the mass of the ions exiting the quadrupole. Ions of a unique m/z ratio pass unobstructed through the quadrupole as a function of the quadrupole power supply (QPS) voltages applied. Ions of different m/z ratios have unstable oscillations that increase in amplitude until they collide with the quadrupole rods and are removed from the ion stream. As an example, a sample mixture containing three molecules, R, M, and N, is introduced into the ion source. Soft ionization in the ion source generates R+, M+, and N+ ions (quasi-molecular ions formed typically by attaching one or more protons in the Positive mode, or by removing one or more protons or attaching an electron in the Negative mode). Isolation of mixture R, M, and N Additional structural information can sometimes be obtained by fragmenting the precursor ion in a primary collision region between the orifice and the skimmer. This process is often referred to as collision induced dissociation mass spectrometry (CID/MS). Isolation of product ions from a sample using the orifice-skimmer technique The ions generated in the ion source are drawn through a curtain of dry inert gas into the ion optics housed inside the vacuum chamber. The mass filter quadrupole in the vacuum chamber selectively filters the ions based on their m/z ratio. The filtered ions are focused to the detector. As ions collide with the detector, they produce a pulse of electrons. The electron pulse is collected and converted to a digital signal to provide an ion count as a function of ion mass. The acquired data is relayed to the computer where it can be displayed as either full mass spectra, intensity of single or multiple ions versus time, or total ion current versus time.

9 Principles of MS/MS In Triple Quadrupole mode, the Q Trap LC/MS/MS system uses two identical mass filter quadrupoles (Q1 and Q3) separated by a collision cell, which encloses an RF-only quadrupole (Q2). The fundamental principle of MS/MS is illustrated in the figure Isolation of product ions from a mixture of R, M and N on page 8. As an example, a sample mixture containing three molecules, R, M and N, is introduced into the ion source. Soft ionization in the ion source generates R+, M+, and N+ ions (quasi-molecular ions formed typically by attaching one or more protons in the Positive mode, or by removing one or more protons or attaching an electron in the Negative mode). Isolation of product ions from a mixture of R, M and N In a Product Ion scan, the first mass filter, Q1, separates or filters ions according to their m/z ratio, and allows only one ion to enter the collision cell (M+). The M+ ion enters Q2 where it is fragmented by collision with neutral gas molecules in a process referred to as collision activated dissociation (CAD). The fragment ions generated are then passed into Q3 and filtered to provide a mass spectrum. The ions created by the source are referred to as precursor ions, the collision products are referred to as product or fragment ions. In a Precursor Ion scan, the third quadrupole (Q3) is fixed to the fragment mass of interest and the first quadrupole (Q1) is scanned over a range. The resulting mass spectrum displays the masses of all the compounds that produced the specified fragment mass. In a Neutral Loss scan, both quadrupoles (Q1 and Q3) are scanned with a constant mass difference between them. The resulting mass spectrum displays the mass of the compounds that have undergone the specified loss. This type of scan is useful in identifying compounds from similar functional groups. The fragment ions are filtered in Q3 before they are collected at the detector. As ions collide with the detector, they produce a pulse of electrons. The pulse is converted to a digital signal that is counted to provide an ion count. The acquired data is relayed to the computer where it can be displayed as either full mass spectra, intensity of single or multiple ions versus time, or total ion current versus time. The technique of MS/MS is well suited to mixture analysis because the characteristic fragment ion spectra can be obtained for each component in a mixture without interference from the other components, assuming that the ions have a unique m/z ratio. This analysis can also be used for targeted analysis by monitoring specific precursor/product ions with Q1 and Q3 respectively while the sample is eluting. This type of analysis is more specific than single MS, which only discriminates on the basis of molecular weight. The MS/MS technique is well suited to structural elucidation studies. The same fragmentation pattern that provides identification of a compound in a complex mixture can also reveal pertinent information regarding the structure of all their precursors. Additional structural information can sometimes be obtained by fragmenting the precursor ion in a primary collision region between the sampling orifice skimmer. The fragment ions (for example, a second generation fragment ion spectrum), provide structural information on both the original precursor ions and the first generation fragment ions. Isolation of second generation product ions from mixture M The triple quadrupole instruments contain the same components as the single quadrupole instruments with the addition of a second mass filter (Q3). The high-pressure region is the same, but the high vacuum region contains the Q1 prefilter (stubbies) and the Q1 and Q3 mass filter quadrupoles that are separated by the collision cell. The collision cell is a ceramic housing enclosing the Q2 RF-only quadrupole, which when pressurized with CAD gas provides a local high-pressure region for ion fragmentation. Ions pass through the same path as in the single quadrupole instrument until they reach the

10 Q2 RF-only quadrupole. The selected ions arrive at Q2, while those rejected eventually collide with the rods and are lost. The Q2 RF-only quadrupole is separated from the Q1 and Q3 mass filters by the interquad lenses IQ2 and IQ3 (or ST3, depending on the triple quadrupole series). The Q2 region has no mass filtering capabilities; it operates in Total Ion mode. If no CAD gas is present to fragment the sample ions, Q2 transports the ions directly into Q3. If CAD gas is present, the ions that enter Q2 collide with the neutral CAD gas molecules. If pressurized, the voltage drop between the entrance lenses and Q2 provides the ions with the energy to induce fragmentation when the ions collide with CAD gas molecules. Through the energetic collisions, the ion translational energy is converted into internal energy that fractures bonds and causes ion fragmentation. After collision, the unfragmented precursor ions and the fragmented ions are transported to Q3 where they are again filtered. When operating in MS/MS mode, the Q3 mass filter is physically and functionally identical to Q1. The ions, including a mixture of precursor and fragment ions, enter Q3 where they are filtered according to mass. In Single MS Operating mode (Q1 scan type), Q3 acts as an ion transporter (like a Q0 or RF-only quadrupole) with no filtering action. Terms used to describe this operation are Total Ion mode, RF-only mode, and AC-only mode. Q Trap LC/MS/MS Enhanced Modes of Operation The Q Trap LC/MS/MS system has a number of enhanced modes of operation. A common factor of the enhanced modes is that ions are trapped in the Q3 quadrupole region and then scanned out to produce full spectrum data. Many spectra are rapidly collected in a short period of time and are significantly more intense than spectra collected in a comparable standard quadrupole mode of operation. The widths of the peaks in the spectra are usually much narrower than peaks observed in the standard quadrupole mode. During the collection phase, ions pass through the Q2 collision cell where CAD gas focuses the ions into the Q3 region. The Q3 quadrupole is operated with only the main RF voltage applied. Ions are prevented from passing through the Q3 quadrupole rod set and are reflected back by an exit lens to which a DC barrier voltage is applied. After the fill time elapses (a time defined by the user), a DC barrier voltage is applied to a Q3 entrance lens (IQ3). This confines the collected ions in Q3 and stops further ions from entering. The entrance and exit lens DC voltage barriers and the RF voltage applied to the quadrupole rods confine the ions within Q3. During the scan out phase, a potential of a few volts is applied to the exit lens to repel the charged ions. An auxiliary AC frequency is applied to the Q3 quadrupole. The main RF voltage amplitude is ramped from low to high values, which sequentially brings masses into resonance with the auxiliary AC frequency. When ions are brought into resonance with the AC frequency, they acquire enough axial velocity to overcome the exit lens barrier and are axially ejected towards the mass spectrometer ion detector. Full spectra data can be acquired from the ions collected in Q3 by rapidly scanning the main RF voltage. The enhanced modes of operation are: Enhanced MS (EMS): Ions are transferred directly from the ion source and orifice region to the Q3 quadrupole where they are collected. These ions are scanned out of Q3 to produce enhanced single-ms type spectra. Use the EMS mode when you need a rapid enhanced sensitivity survey type scan. Enhanced Resolution (ER): This mode is similar to the Enhanced Product Ion mode

11 except that the Q1 precursor ions pass gently through the Q2 collision cell without fragmenting. A small range about the precursor mass is scanned out of Q3 at the slowest scan rate to produce a narrow window of the best-resolved spectra. Enhanced Product Ion (EPI): Product ions are generated in the Q2 collision cell by the precursor ions from Q1 colliding with the CAD gas in Q2. These characteristic product ions are transmitted and collected in Q3. These ions are scanned out of Q3 to produce enhanced product ion spectra. Use the EPI mode if you need enhanced resolution and intensity. Enhanced Multi-Charge (EMC): This mode operates similarly to the Enhanced MS mode except, before scanning the ions out of Q3, there is a delay period in which low charge state ions (primarily singly charged ions) are allowed to preferentially escape from the Q3 quadrupole. When the retained Q3 ions are scanned out, the multiply charged ion population dominates the resulting spectra. Time Delayed Fragmentation (TDF): Product ions are generated and collected in Q3. During the first part of the collection period, the lower mass ions are not collected in Q3. During the second part of the collection period, all masses over the mass range of interest are collected. The resultant enhanced product ion spectra are simplified compared to EPI scan type spectra. The nature of the spectra aids in the interpretation of the structure and fragmentation pathways of the molecule of interest. MS/MS/MS (MS3): In MS/MS/MS mode, product ions are generated in the Q2 collision cell by the precursor ions from Q1 colliding with the CAD gas in Q2. These characteristic product ions are transmitted and collected in Q3. Applying the normal mode resolving DC voltages to the Q3 quadrupole isolates a specified mass (m/z) of ion and removes all other ions from Q3. By properly applying a second auxiliary AC frequency to Q3, the specified ion can be resonantly excited. These excited ions collide with the residual nitrogen in Q3 and may fragment, producing a characteristic spectrum of ions. These secondary product ions of the isolated product ion result in MS/MS/MS product spectra. Proudly serving laboratories worldwide since 1979 CALL for Refurbished & Certified Lab Equipment