MALDI-TOF/TOF MS Protocols

Transcription

1 MALDI-TOF/TOF MS Protocols Objectives: 1. To be familiar with the basic operation of the MALDI-TOF-TOF mass spectrometer. 2. To use MALDI-TOF-TOF mass spectrometer for peptide mass fingerprinting, peptide sequencing, and in source decay sequencing of intact proteins. Protocols included: 1. Obtaining a peptide mass fingerprint with MALDI-TOF 2. ISD and T 3 sequencing 3. In-gel tryptic digest 4. Sample spotting with the dried droplet method 5. Ultraflex III Operation for Obtaining a PMF Introduction: MALDI refers to the ion source, i.e., the Matrix Assists in Laser Desorption/Ionization of the analyte. The matrix, is co-crystallized with the sample, it must absorb light maximally at 337 nm (wavelength of the laser), and must participate in proton transfer with the analyte. TOF refers to the mass analyzer. The mass analyzer separates ions based on their Time Of Flight. Flight time is a function of the mass to charge ratio (m/z) of the analyte. TOF-TOF means that this is a tandem mass spectrometer, so an ion can be selected for, fragmented, and mass analyzed again. Tandem mass spectrometry, MS/MS, can provide additional information for structural elucidation of the analyte in question. The Ultraflex III is a MALDI-TOF-TOF mass spectrometer that will be used in this laboratory (Figure 1). A suite of software is used with this instrument. Acquisition of mass spectra is done in Flex Control, spectrum processing and annotation in Flex Analysis, and additional data processing and data searching is done in BioTools and Sequence Editor (Bruker). 1

2 Figure 1: Schematic of the Ultraflex III MALDI-TOF-TOF. ( Bruker training presentation) A typical peptide mass fingerprint (PMF) work flow would involve digesting a target protein with trypsin, obtaining a mass spectrum of the peptides, and using those m/z values to search the databases for a match. This method relies solely on the peptide map obtained in the mass spectrum and not on the amino acid sequence. If cross-species identification is used during the data search, the sample must have sequence identity for a subset of the protein. For proteins with some post translational modification, a PMF can help with identifying those regions, but too many post translational modifications may result in no identification. Reviews are available (Gilany, et al. 2010; Henzel, et al. 2003). A significant PMF result requires a mass tolerance of approximately 1 Da and in a MALDI-TOF PMF experiment, a mass accuracy of better than 20 ppm (0.02 Da for an m/z of 1000) can be obtained. The mass spectrum is acquired as follows. The cocrystallized matrix-analyte is hit with the laser. Irradiation of the matrix and analyte causes a plume to form in which proton transfer occurs between the matrix and analyte, resulting in ionization. After a delay, the ions are accelerated by an electric field towards the TOF mass analyzer where ions of differing m/z are separated based on their flight time. This flight time is converted to mass values, which are often in the form of isotope patterns, due to the high resolving power of the instrument. This allows for determination of the exact mass and structural information. Resolution can be improved in several ways. Each way involves decreasing the velocity distribution of ions having the same m/z. First, the matrix-analyte crystals should be a flat layer. This in effect allows ions to have an equal starting point. The ion source is equipped with pulsed ion extraction, or delayed extraction of ions. After the plume of ions is formed, ion source plate 1 (IS/1) and ion source plate 2 (IS/2) are kept at the same voltage for a short period of time, allowing slower ions to catch up to the faster ions. After this delay, the voltage of IS/2 is dropped and ions are accelerated towards the detector. Adjustment of the delay time, lens voltage, and digitizing rate of the detector can improve the sensitivity and resolution of some analytes. 2

3 Figure 2: Pulsed ion extraction. (Bruker training presentation.) Operating in linear mode has the advantage of increasing sensitivity and is often used for molecular weight determination of proteins. Operating in reflector mode, while decreasing the sensitivity, drastically improves the resolution. Instead of hitting the linear detector, the ions are reflected in an ion mirror. This increases the flight time, and focuses the ions. The results of a PMF can be strengthened or confirmed through peptide sequencing. Tandem mass spectrometry can be performed in two ways with the Ultraflex III: laser induced dissociation (LID) for peptide mass fingerprinting, or collision induced dissociation (CID) for de novo sequencing because the high energy collisions with Argon gas produce fragment ions used in distinguishing between isobaric residues. The laser power is increased relative to MS so that more precursor ions per shot are obtained. The initial accelerating voltage is low relative to MS, allowing for a long flight time (10-20 μs). During this time, ion families are formed through metastable decay of parent ions. The ion family of interest passes through a voltage gate, the timed ion selector, while other ion families are deflected. The ion family is post-accelerated and focused in the lift cell. The post-lift metastable suppressor acts like the timed ion selector and deflects parent ions while fragment spectra are acquired. After focusing by the ion reflector, ions are detected by the reflector detector (Suckau et al. 2003). Peptide mass fingerprinting and identification by peptide sequencing are considered as bottom-up approaches. A top-down approach is available, where intact proteins are identified based on the sequencing of their N- and C-termini. ISD sequencing is performed as a MS experiment in positive reflector mode, using a method optimized for peptides. Instrument parameters are adjusted so that fragmentation of the termini can occur in the plume before ion extraction. The acceleration voltage is 25 kv, pulsed ion extraction time set at 200 ns laser power and the detector gain are increased. Unambiguous sequence assignment is attributed to the predictable fragmentation along the peptide backbone. Fragments from the N- terminus are predominantly c-ions, and fragments from the C- terminus are y- ions. ISD sequencing cannot be used for ions below 1000 m/z since this region of the mass spectrum is dominated by matrix peaks. T 3 sequencing, i.e., MS/MS of an ISD fragment ion, is performed for sequencing of the 3

4 terminal residues (Suckau and Reseman, 2003). Figure 3 shows a schematic of this approach. Processing methods perform peak picking, baseline subtraction, and smoothing operations. During peak picking, signal to noise ratio, relative intensity threshold, minimum intensity threshold, and maximum number of peaks are all taken into account. Three peak picking algorithms are available: SNAP, centroid, and sum. SNAP (Sophisticated Numerical Annotation Procedure) searches for isotope patterns in the spectrum and performs baseline correction and noise determination. The result is that the monoisotopic peak is identified for each isotope pattern. Identifying the monoisotopic peak is necessary for obtaining a PMF. are faster peak picking algorithms. Centroid and Sum calculations do not take the isotope pattern into consideration for peak picking. Centroid finds peaks based on first and second order derivative calculations. Centroid is used for proteins and the average peak, as opposed to the monoisotopic peak is calculated. The Sum algorithm is used in applications where speed is a key factor; peaks are picked based on a pseudo-derivative calculation. For Centroid and Sum methods, baseline subtraction and smoothing operations can be adjusted by the user as needed. Figure 3: ISD fragmentation provides the amino acid sequence near the termini. Each ISD fragment contains the actual terminus so one of these ions can be used to obtain the sequence of the terminus (Suckau and Reseman, 2003). The Mascot search engine (Matrix Science) is used to search available databases during protein identification experiments. The first step in PMF searching is to define the search. Figure 4 shows a search dialog box. Close attention should be paid to taxonomy, database, variable modifications, and mass tolerance. These search parameters can drastically affect the search results. Points on some of the search parameters are listed below. 4

5 Database: Some databases (e.g., SwissProt) are highly annotated and contain few errors, but are limited on the number of entries. Others (e.g. expressed sequence tag (EST) databases) have many more entries but contain errors, redundancies, and entries are not annotated. Variable vs. global modifications: Global refers to the known chemistry of the sample while variable refers to possible modifications. Missed cleavages: This refers to the completeness of the digest. For example, the tryptic peptide, GHMNIRFR, contains 1 missed cleavage. Mass tolerance: ppm is generally used for a MALDI-TOF PMF. If the mass tolerance is too restricted, the search may not result in a significant assignment. Data file or query data: This is automatically entered if going through BioTools. If searching from the Matrix Science website, it is entered manually. Mascot search results are given as a histogram of the top scores. Hits in the green region have a higher chance (5% or more) of being a false positive and hits outside of this region (more than 70) are considered significant. The score for the false positive likelihood is a based on the search conditions and the database used. Figure 4: Mascot search box for peptide mass fingerprinting. 5

6 The format of the results can be adjusted with the Format As dropdown box. The results can be viewed as groups of homologs, in an index, coverage of a specific protein, or as an error plot. The error is shown as the root mean square (RMS) error, a measure of the average mass error of the dataset. Figure 5: A histogram of the Mascot search results. Protocols 1. Obtaining a peptide mass fingerprint with MALDI-TOF Sample spotting: Spot the sample with α-cyano-4-hydroxycinnamic acid (HCCA) using the dried droplet method. The peptide digest should be at a concentration of 1 pmol/µl. Spot Peptide Mix II calibration standard. Acquisition method: RP_Proteomics_HPC is the FlexControl method. External calibration is performed with peptide calibration mix II. Data processing and analysis: The Protein Mass Fingerprint Flex Analysis Mass Spectrometry (PMF.FAMS) method, which uses the SNAP peak picking algorithm, is used for spectrum processing. Mascot search is performed from BioTools. Peptide sequencing with MALDI-TOF-TOF Sample spotting: Spot the sample with HCCA using the dried droplet method. The peptide digest should be at a concentration of 1 pmol/µl. No calibration standard is necessary. (Calibration is performed for the LIFT method approximately once a year with a different method than what is described here). Acquisition: A current LIFT method is used in FlexControl. Data Processing and analysis: SNAP_full_process method in FlexAnalysis is used for processing. The sequence can be annotated manually in Flex Analysis or a Mascot search can be performed in BioTools. 2. ISD and T 3 sequencing 6

7 Sample spotting: Use the dried droplet method to spot the pure, intact protein ( pmol/µl). 2,5-Dihydroxybenzoic acid (DHB) is recommended. ISD acquisition: In Flex Control, a peptide method is used. The low mass deflection set to 900 Da, detection gain to 20X, and pulsed ion extraction set to 200 ns. ISD data processing and analysis: The spectrum is processed in Flex Analysis with the PMF_FAMS method, and then it is sent to BioTools In the Analysis pull down menu, select Permanently assign as an ISD type 2. Switch to MS/MS analysis and choose Top Down 3. Click on Find Tags 4. Settings can be changed, but default settings are usually a good starting point. Click Create Sequence Tags 5. Sequence tags are listed in order of highest to lowest ranking. They are ranked based on Σ (peak area/number of peaks). The top five are automatically highlighted. Click Copy/MS BLAST, then Open MS BLAST, and paste into the provided field. Click Submit Query. 6. Press Open Hit in SE, and Sequence Editor will open with the sequence. 7. Truncate the sequence to send it to BioTools. ISD is used to sequence the termini of the protein, so use the first 100 of each terminus. Send these sequences to BioTools. 8. In BioTools, change the annotation parameters to ISD_extended. Work in the MSMS Fragments tab to check the mapping of the theoretical mass fragments to those in the experimental mass spectrum. T 3 sequencing acquisition: In Flex Control, use a LIFT method. Define an ISD fragment ion as the parent, detection gain may need to be increased and several thousands of shots are likely to be required since sensitivity is low for this method. Data processing and analysis: In Flex Analysis, process the spectrum with SNAP_full_process. The sequence can be annotated manually in Flex Analysis or a Mascot search can be performed in BioTools using Amide (C-term) as the variable modification. 3. In-gel tryptic digest Materials 55 mm iodoacetamide: Dissolve 10.2 mg of IAA to a final volume of 1.0 ml with 100 mm ammonium bicarbonate. 100 mm ammonium bicarbonate: Dissolve g of NH 4 HCO 3 in 50 ml with HPLC grade H 2 O. Dehydration solution (HPLC grade acetonitrile) Peptide extraction solution (50% acetonitrile, 5% formic acid) Protein sample embedded in gel plug 10 mm dithiothreitol (DTT): Dissolve 1.54 mg of DTT to a final volume of 1.0 ml with 100 mm ammonium bicarbonate. Trypsin solution (Promega sequencing grade modified trypsin (Cat. # V5111): One vial contains 20 μg of trypsin. Dissolve in 200 μl of reconstitution buffer (50 mm

8 acetic acid) provided with trypsin. Aliquot the trypsin into 50 μl aliquots and freeze at -80 C. Trypsin is inactive at ph 4.0 or below. Scale this so that the trypsin solution is only re-frozen less than 5 times. Procedure 1. Cut protein band from the gel as close to the stained band as possible. Place in an eppendorf tube and remove any excess liquid. 2. For gels stained with Coomassie Blue, destain the plug by dispensing 50 μl of 50 mm ammonium bicarbonate and 50 μl of acetonitrile. Incubate for 10 minutes at 37 C. Aspirate the solution and discard. Repeat until gel plug is clear. 3. Dehydrate the plug in 50 μl acetonitrile for 5 minutes at 37 C. Aspirate the solution, discard, and incubate the gel plug for 10 minutes at 37 C. It should turn opaque-white. 4. Reduce the protein in 50 μl of 10 mm DTT, for 20 minutes at 37 C. Aspirate the solution and discard. 5. Alkylate the protein in 30 μl of 55 mm iodoacetamide for 20 minutes at 37 C. Aspirate the solution and discard. 6. Dehydrate the gel plug (see step 3). 7. Activate the trypsin solution. Add 160 μl of 50 mm ammonium bicarbonate to a 50 μl aliquot of trypsin (aliquot is 100 μg/ml). Thus, 15 μl will contain 300 ng of trypsin. (The addition of ammonium bicarbonate activates the trypsin, which is maximally active in the ph range 7-9). 8. Add 15 μl of the active trypsin solution to the gel plug and incubate at room temperature for 10 minutes to allow the gel plugs to swell. 9. Add 15 μl of 50 mm ammonium bicarbonate and incubate for 4-16 hours at 37 C. 10. Add 40 μl of peptide extraction solution to halt digestion and extract peptide. Sonicate 2 times for 5 minutes each. Combine extracts in a tube. 11. Evaporate solution under SpeedVac for 1 h at RT. 12. Add 50 μl of 5 % formic acid to dissolve the peptide residue. Or add 0.1% triflouroacetic acid (TFA). 4. Sample spotting with the dried droplet method Materials Matrix (20 g/l HCCA (α-cyano-4-hydroxycinnamic acid), DHB (2,5-dihydroxybenzoic acid), or SA (sinnapinic acid) in acetonitrile: 0.1% trifluoroacetic acid (70:30 v/v)) Peptide/protein sample in 0.1% trifluoroacetic acid Calibration standard (PepmixII, ProtMixI or ProtMixII stored at -20 C in aliquots in 0.1% TFA) Ground Steel Target Plate Procedure 1. Dissolve HCCA. Sonicate for 5 minutes, then centrifuge for 5 minutes. 2. Apply 0.5 μl of the sample solution to a target spot. Overlay with 0.5 μl of the binary matrix and mix. Prepare, as in step 3, a calibration spot near the sample. 3. Allow samples to air dry or dry them under a gentle stream of N 2 gas. 8

9 5. Ultraflex III Operation for Obtaining a PMF 1. Turn on computer. 2. Double-Click Flex Control icon to open acquisition program. 3. Insert the target plate/holder into the instrument. First, press the bottom of the door, open the latch and gently place the target into the carrier. The correct orientation is for the 1 column and A row to be in the top left corner with the sample side facing towards the entrance to the MS room. 4. In Flex Control, you are prompted to select a Method. Choose the most recent RP_Proteomics_HPC method. (Reflector detector, Positive polarity) 5. Single-Click the Inject/Eject icon to insert the target. 6. Go to the Details tab. Record the number of laser shots taken so far in the instrument log book. 7. When the target is inserted, Single-Click on the spot containing the calibration standard. The spot will now be shown in the camera view. The cross-hairs designating where the laser will shoot will be in the center of the spot. 8. Go to the Calibration tab. From the dropdown list, select the calibrant Peptide Calibration Standard II Monoisotopic. 9. Decrease the laser intensity to about 5%. With the cursor in the vicinity of the camera viewer, the wheel can be used to change the laser intensity. Click Start and slowly increase the laser intensity until the calibration standard peaks can be seen. Once this laser intensity has been identified, increase laser a little past this threshold. Do not increase if the baseline drifts upward. This is a sign that laser intensity is too high. Excessive noise and low signal intensity is a sign that laser intensity is too low. 10. After the spectrum has been acquired, click Add. Several points of acquisition can be added to the sum buffer. To get rid of an acquisition point that has been added, click Undo. To get rid of the summed acquisitions, click Clear Sum. 11. Go to the Calibration tab. Click Automatic Assign. The mass tolerance is set to 500 ppm and uses a quadratic function by default. The automatic assign function will select the monoisotopic peaks in the spectrum that correspond to the expected m/z values. For it to be a good calibration, the standard deviation should be within +/- 5 ppm of the expected value. 12. For the calibration to take effect, click Assign. Then go to File à Save Method As and save the method under a new name, in your folder. This personal method can be saved over each time you calibrate. The default method cannot be saved over. The default method should be used each time you calibrate. 13. Click on the spot containing the analyte of interest using the Target Map Icon. Decrease the laser intensity again to about 5%. Click Start and slowly increase the laser intensity until you see the analyte signal. Click Add and collect about 400 laser shots in total. 14. Click Save As. You will be prompted to write any relevant notes, where the file should be saved to, and under what name. Put a tick mark in the box for Open in Flex Analysis. In the dropdown box for Processing Method choose PMF.FAMS. Click Save. 15. The window for Flex Analysis will automatically open. The summed spectra that were acquired will be processed and annotated within the mass range of m/z. 9

10 16. To eliminate background peaks, click on MassList/Filter Background Peaks. Choose the MassControlList appropriate to the sample. For example, trypsin autoproteolysis or contaminants like keratins. 17. Press the icon for Biotools. The processed spectrum will be sent to the Biotools program. 18. Press the icon MS to open the MASCOT search window. 19. Enter all relevant information into the MS Search Dialogue. 20. Click Start and wait for the MASCOT results. 21. To export the top hit from the MASCOT Search Results window, click Get Hits. Close the window. The results will be displayed in BioTools. 6. References Gilany, K., Moens, L., & Dewilde, S. (2010). Mass spectrometry-based proteomics in the life sciences: a review. Journal Of Paramedical Sciences, 1(1). Henzel, W. J., Watanabe, C., & Stults, J. T. (2003). Protein identification: the origins of peptide mass fingerprinting. Journal of the American Society for Mass Spectrometry, 14(9), Link, A. J. and LaBaer, J. (2009). Proteomics: A cold Spring Harbor Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Suckau, D., Resemann, A., Schuerenberg, M., Hufnagel, P., Franzen, J., and Holle, A., A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Analytical and Bioanalytical Chemistry, 376(7): , August Suckau, D. and Resemann, A., T 3 -Sequencing: Targeted Characterization of the N- and C-termini of Undigested Proteins by Mass Spectrometry. Analytical Chemistry, 75(21): , November In-house protocol for protein analyses by LC-IT-MS Tutorials for BioTools, Version 3.1 (January 2007) 10