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MALDI-TOF/TOF MS Protocols Objectives

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MALDI-TOF/TOF MS Protocols Objectives 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 T3 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 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 co- crystallized 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 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. T3 sequencing, i.e., MS/MS of an ISD fragment ion, is performed for sequencing of the 3 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 ● 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: 20 – 50 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 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.
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