MALDI-TOF-TOF MS协议参考资料.pdf

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 3 2. ISD and T 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