The document discusses MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. It provides a history of the development of mass spectrometry techniques. MALDI-TOF allows for the analysis of intact biomolecules like proteins and is a soft ionization method. It provides high sensitivity and mass accuracy for analyzing proteins, peptides, and other large biomolecules. The document also discusses applications of MALDI-TOF like protein identification and characterization.
MALDI-TOF: Pricinple and Its Application in Biochemistry and Biotechnology
1. MALDI-TOF: PRINCIPLE & APPLICATIONS C. Devakumar Division of Agricultural Chemicals IARI, New Delhi [email_address]
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3. The ability to separate molecules based on different size and charge was first described in 1912 by J.J. Thompson (Nobel Prize laureate in 1906 for investigations of the conduction of electricity by gases) and expressed as the mass/charge ratio with the unit Thompson (Th). M.S.B. Munson and F.H. Field in 1966, made early major breakthrough in the use of chemical ionisation (CI). Plasma desorption (PD), introduced in 1976, uses high-energy ions to desorb and ionise molecules. The technique achieved some success but was never shown to be reliable for molecular masses greater than 10 kiloDalton (kDa). Fast atom bombardment (FAB), and the closely related method liquid matrix secondary ion mass spectrometry (LSIMS) used Accelerated atoms (and later also ions) of e.g. argon, caesium or xenon could be used for mass determination of small biomolecules (i.e. mol. wt. <10 kDa) combined with on-line fragmentation for structure determination. . HISTROY OF MASS SPECTROMETRY
6. The well-defined breakthrough of ESI came in 1988 at a symposium in San Francisco, when John Fenn presented an identification of polypeptides and proteins of molecular weight 40 kDa. Fenn showed that a molecular-weight accuracy of 0.01% could be obtained by applying a signal-averaging method to the multiple ions formed in the ESI process. Matrix-assisted, laser-desorption ionisation (MALDI) technique applied to proteins appeared shortly after Tanakaās initial breakthrough. The MALDI technique presented by M. Karas and F. Hillenkamp used a YAG laser at 266 nm and a chemical matrix of nicotinic acid DISCOVERY OF MALDI
16. Voyager-DE STR MALDI TOF Camera Laser Sample plate Pumping Pumping Timed ion selector Reflector Linear detector Extraction grids Reflector detector Attenuator Prism Collision cell
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21. MALDI is also a "soft" ionisation method and so results predominantly in the generation of singly charged molecular-related ions regardless of the molecular mass, hence the spectra are relatively easy to interpret. Fragmentation of the sample ions does not usually occur although they can be accompanied by salt adducts, a trace of the doubly charged molecular ion at approximately half the m/z value, and/or a trace of a dimeric species at approximately twice the m/z value.. In positive ionisation mode the protonated molecular ions (M+H + ) are usually the dominant species, It is used for protein and peptide analyses. In negative ionisation mode the deprotonated molecular ions (M-H - ) are usually the most abundant species, and can be used for the analysis of oligonucleotides and oligosaccharides .
22. Works in the range of molecular masses between 400 and 350,000 Da. A very sensitive method, the detection of low (10 -15 to 10 -18 mole) quantities of sample with an accuracy of 0.1 - 0.01 % with short measuring time (few minutes) and negligible sample consumption (less than 1 pmol) together with additional information on microheterogeneity (e.g. glycosylation) and presence of by-products. The mass accuracy of MALDI-TOF MS will be sufficient to characterise proteins (after tryptic digestion) from completely sequenced genomes.
23. MALDI-TOF MS analysis of natural pruducts Chlorophylls lipids and glycolipids folic acids storage products mycotoxins pigments alkaloids siderophores cyanobacterial peptides food ingredients polymers DNA and RNA and proteins directly from whole cells and samples without purification steps.
30. MALDI-TOF mass analysis of the peptide mixture, database searches and protein identification: Analysing peptides from protein digests to identify the protein. A band or spot can be cut from a 1D or 2D gel, the protein digested in-gel , and after Zip-Tip cleanup , the peptides are analyzed with the mass spectrometer.
31. Sample Clean up Using Zip-Tips in Preparation for MALDI-TOF Mass Analysis: Zip-Tips are pipette tips that contain immobilized C18, C4 or some other resin attached at their very tip occupying about 0.5Āµl volume. The usual protocol is: Use a P20 pipetter set to 10Āµl for Zip-Tips Wash the Zip-Tip with 0.1% trifluroacetic acid (TFA) in acetonitrile Wash the Zip-Tip with 0.1% TFA in 1:1 acetonitrile:water quilibrate the Zip-Tip twice with 0.1% TFA in water The sample, dissolved in 10 Āµl of 0.1% TFA, is passed through the Zip-Tips repeatedly by pipeting in and out to bind the sample to the resin. Wash the Zip-Tip three times with 0.1% TFA, 5% methanol in water Elute the sample from the Zip-Tip in 1.8Āµl of matrix, typically alpha-cyano-4-hydroxycinnamic acid in 0.1% TFA 50% acetonitrile, directly on the MALDI-TOF sample plate.
33. Peptide Mass Fingerprinting: The sample plate with up to 100 spots is inserted in the mass spectrometer A laser is applied to individual spot thus ionizing molecules of the matrix which in turn transfers a proton to the peptides. Peptides are accelerated through the flight tube under vacuum and in most cases in a reflector mode, which basically makes the flight path longer than the actual tube. Peptides arrive at the detector based on their mass to charge ratio (m/z). Using calibration peptides, the actual masses of the peptides are assigned.
35. Data base search and Protein Identification: All the masses that represent peptides from the original protein (in other words, masses present in control samples where no protein was present are ignored) represent the fingerprint of the protein in question. By searching a mass database for protein fingerprints, the protein is identified if known. If we are dealing with an unknown protein, further identification becomes necessary among which is peptide sequencing of selected peptides by post source decay (PSD) or Collision induced dissociation (CID).
39. Advantages: Rapid analysis and turn around time High sensitivity Cheap Suitable for large numbers of samples Disadvantages: Protein must be in the database Generally not suitable for proteins <15kDa in size Match based on peptide masses, not sequence information Generally only able to suggest post-translational modifications
40. Protein Identification by MALDI-TOF/TOF (PMF + MS/MS) Proteins are digested in the same manner as for peptide mass fingerprinting and the sample is then analysed by MALDI-TOF, generating a peptide mass fingerprint for the protein. The most abundant peptide ions are then subjected to MALDI-TOF/TOF analysis, providing information that can be used to determine the sequence. The results from both types of analysis are combined and searched using software (e.g. Mascot) against protein, DNA or EST databases, to identify the protein.
41. Advantages: Rapid analysis and turn around time (similar to MALDI-TOF)Ā High sensitivityĀ Relatively inexpensiveĀ Suitable for large numbers of samplesĀ Able to identify 2-3 proteins in the same spotĀ Sequence information provides confirmation of peptide mass fingerprint identification & allows identification of small proteins (<15kDa) Disadvantages: Sequence information generally not as complete as that provided by LC/MS/MSĀ Limited success in identification of proteins that are not in the database
43. MALDI/TOF/TOF MS glycomic profile of permethylated N - and O -glycans derived from human blood serum. Symbols: ā , N -acetylglucosamine; ā, mannose; ā”, galactose; ā, fucose; āµ , N -acetylneuraminic acid.
44. LC/MALDI/TOF/TOF MS of online permethylated glycans derived from a mixture of glycoproteins. Symbols: ā , N -acetylglucosamine; ā, mannose; ā”, galactose; ā, fucose; āµ, N- acetylneuraminic acid.
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46. Expression, Purification, and Characterization of C-Terminal Amidated Glucagon in Streptomyces lividans Qi, Xiaoqiang, Rong Jiang, Cheng Yao, Ren Zhang, and Yuan Li Glucagon, a peptide hormone produced by alpha-cells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, its structure was modified at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat Ī±-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans . With Phenyl-Sepharose 6 FF, QSepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the N-terminal part. J. Microbiol. Biotechnol . (2008), 18(6), 1076ā1080
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48. 2. The Sensitive Type The devil is always in the nitty-gritty details, and for proteins, that means post-translational modifications. Suppose, for instance, that you're looking at histones, which can bear both acetyl and trimethyl modifications. Both moieties produce nominal mass increases of 42, and a standard mass spec cannot distinguish the two. A high-mass-accuracy instrument can, however, since it can report masses to between two and four decimal places. Recommended System: LC+ESI+FTICR with ECD
49. 3. The Outsider Not everyone is interested in proteins. You might want to know, for instance, if a particular nucleic acid contains unusual or modified residues (such as methyl-C), and if so, where in the sequence they are located. Both questions may be addressed using an LC-ESI-tandem mass spec (such as a QTOF or Qtrap configuration); the former in negative-ion mode (because of the nucleic acid's negatively charged backbone), and the latter in positive mode.
50. 4. The Mixer Sifting through small-molecule metabolites (sugars and lipids, for example) requires a different set of instrumentation considerations. You might be operating in discovery mode, looking for a biomarker for a particular disease, say, or drug efficacy. In that case, you'll need tandem mass spec capabilities to nail down chemical structure, and your instrument of choice is an LC-ESI-triple quad. More importantly, however, you'll need multiple ionization methods to cast the widest net. Consider using the two electrospray variants, APPI (atmospheric pressure photoionization) and APCI (atmospheric pressure chemical ionization). Recommended System: LC+ESI+triple quad with multiple ionization sources
51. 5. The Counter Once you've identified your biomarker, you now need to count it, perhaps in hundreds or thousands of biological samples. The go-to mass analyzer for quantitative applications is the triple quad, which you'll want to couple to liquid chromatography and an electrospray ionization source.
52. PerkinElmer's prOTOF 2000 MALDI-TOF employs a new orthogonal geometry It can hold its calibration for a minimum of one hour, It eliminates the problem of ion suppression, even if the sample is at a lower concentration than the internal standard. Employs single-use, disposable targets called MALDIchipsā¢, which are available in 96, 384, and 1536-sample formats compatible with standard liquid handling devices
53. Applied Biosystems new 4800 MALDI-TOF/TOF Analyzer In the new system, the laser beam is perpendicular to the target plate, so the resulting column of ions reflects back along the laser's axis and directly down the flight tube. Another improvement is the QuanTIS timed precursor ion selector, which isolates a specific precursor ion from the first TOF run for analysis in the second TOF analyzer. $485,000