maldi-tof: pricinple and its application in biochemistry and biotechnology
DESCRIPTION
This is a lecture given in the Centre of Advanced Studies, Division of Biochemistry, Indian Agricultural Research Institute, New DelhiTRANSCRIPT
MALDI-TOF: PRINCIPLE & APPLICATIONS
C. DevakumarDivision of Agricultural Chemicals
IARI, New [email protected]
• Biological macromolecules are the main actors in the make-up of life whether expressed in prospering diversity or in threatening disease.
• To understand biology and medicine at molecular level where the identity, functional characteristics, structural architecture and specific interactions of biomolecules are the basis of life, we need to visualize the activity and interplay of large macromolecules such as proteins.
• To study, or analyse, the protein molecules, principles for their separation and determination of their individual characteristics had to be developed.
BIOCHEMISTRY = CHEMISTRY OF LIFE
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
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 Tanaka’s 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
UV MALDI Matrix List
CompoundOther Names Solvent
Wavelength (nm)
Applications
2,5-dihydroxy benzoic acid
DHB, Gentisic acid
acetonitrile, water, methanol, acetone, chloroform
337, 355, 266
peptides, nucleotides, oligonucleotides, oligosaccharides
3,5-dimethoxy-4-hydroxycinnamic acid
sinapic acid; sinapinic acid; SA
acetonitrile, water, acetone, chloroform
337, 355, 266
peptides, proteins, lipids
4-hydroxy-3-methoxycinnamic acid
ferulic acidacetonitrile, water, propanol
337, 355, 266
proteins
α-cyano-4-hydroxycinna
mic acidCHCA
acetonitrile, water, ethanol, acetone
337, 355peptides, lipids, nucleotides
Picolinic acid PA Ethanol 266oligonucleotides
3-hydroxy picolinic acid
HPA Ethanol 337, 355oligonucleotides
Lasers Used for MALDI
LaserWavelength
(nm)Reference
Nitrogen laser
337 (Tanaka 1988)
Nd:YAG 355, 266 (Karas 1985)
Er:YAG 2940(Overberg 1990)
CO2 10,600(Overberg 1991)
Time-of-flight mass analyzer
+
+
+
+
Source Drift region (flight tube)
dete
ctor
V
•Ions are formed in pulses.
•Small ions reach the detector before large ones.
•Measures the time for ions to reach the detector.
Voyager-DE STR MALDI TOF
Camera
Laser
Sample plate
Pumping Pumping
Timed ion selector Reflector
Linear detector
Extractiongrids
ReflectordetectorAttenuator
Prism
Collision cell
MALDI TOF HardwareLaser, Attenuator and PrismNitrogen laser at 337 nm, 3 ns wide pulses, 20 Hz. Laser attenuator varies the intensity of the laser hitting the sample. Prism deflects the laser beam into the ion source.Sample Plate and Sample StageAn accelerating voltage is applied to the sample plate in the range 15-25 kV.Variable Voltage GridA grid 1-2 mm above the sample plate with an additional voltage to fine- tune ion accelerationGround GridGrounded surface defines end of acceleration regionGrounded ApertureEntrance to flight tube
MALDI TOF Hardware
Vacuum System
High vacuum is required to avoid ion collisions
Flight tube
A field free region where ions drift at a velocity inversely proportional to the square root of their mass/charge.
Linear Detector
Measures the ion abundance in linear mode (no reflector used) and sends a signal to the digitizer.
MALDI TOF HardwareReflectorA single stage gridded ion mirror that subjects the ions to a uniform repulsive electric field to reflect them. It is tilted by 1° in the DE-STR to focus the ions on to the detectorCollision CellGas cell for collision induced dissociation (CID) to enhance fragmentation in PSD analysisReflector DetectorMeasures ions reflected by the mirror. In the DE-STR this is a 6-10 m pore size micro-channel plate. Timed Ion SelectorA velocity selector that allows a single precursor ion of a selected mass and their fragment ions to pass to the detector. A Bradbury-Neilson gate is used.
Features of MALDI-TOF MS
• Soft ionization - analyze intact biomolecules and synthetic polymers
• Broad mass range - analyze a wide variety of biomolecules
• Simple mixtures are okay
• Relatively tolerant of buffers and salts
• Fast data acquisition
• Easy to use and maintain, no water or gas hook ups required
• High sensitivity, superior mass resolution and accuracy
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.
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.
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.
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.
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 0.5µl volume. The usual protocol is: Use a P20 pipetter set to 10µl 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 µl 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 1.8µl of matrix, typically alpha-cyano-4-hydroxycinnamic acid in 0.1% TFA 50% acetonitrile, directly on the MALDI-TOF sample plate.
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.
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).
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
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.
Advantages:Rapid analysis and turn around time (similar to MALDI-TOF) High sensitivity Relatively inexpensive Suitable for large numbers of samples Able to identify 2-3 proteins in the same spot 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/MS Limited success in identification of proteins that are not in the database
MALDI/TOF/TOF MS glycomic profile of permethylated N- and O-glycans derived from human blood serum.
Symbols: ■, N-acetylglucosamine; ○, mannose; □, galactose; ●, fucose; ▵, N-acetylneuraminic acid.
LC/MALDI/TOF/TOF MS of online permethylated glycans derived from a mixture of glycoproteins.
Symbols: ■, N-acetylglucosamine; ○, mannose; □, galactose; ●, fucose; , ▵ N-acetylneuraminic acid.
Comparing ionization techniques• MALDI
– Bias for olar/charged peptides– Relatively salt tolerant– Suitable for complex mixes– Must be “offline”– Relatively low res due to
desorption velocity– Matrix <1000 m/z– Photo crosslinking and
degradation– Nobel Prize in Chemistry,
2002
ESI
– Bias for nonpolar peptides.
– Very salt sensitive
• Low tolerance for very complex mixes– Requires expertise and
$’s to run and maintain– Analyses can be coupled
to µLC– > sequence coverage– Nobel Prize in Chemistry,
2002
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 α-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), 1076–1080
1.The Down and Dirty Protein Chemist
The protein chemist just wants to know what it is that he's looking at. He could be looking to understand the biology of a protein by characterizing its coimmunoprecipitation partners, or identifying a particular spot on a two-dimensional gel. For these kinds of applications a quick and dirty approach will generally give the answers he needs.
Recommended System: MALDI+TOFSimple and fast
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
3. The OutsiderNot 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.
4. The MixerSifting 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
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.
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 MALDIchips™, which are available in 96, 384, and 1536-sample formats compatible with standard liquid handling devices
Applied Biosystems new 4800 MALDI-TOF/TOF AnalyzerIn 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