A mass spectrometer determines the mass of a molecule by measuring the mass-to-charge ration (m/z) of its ion. Ions are generated by inducing either the loss or gain of a charge from a neutral species. Once formed, ions are electrostatically directed into a mass analyzer where they are separated according to m/z and finally detected. The result of molecular ionization, ion separation, and ion detection is a spectrum that can provide highly accurate molecular mass and mass structural information.
An analyte is a collection of peptides derived protein after digestion. Two types of analyses are carried out on an analyte:
• Analysis of intact peptide ions - PMF
• Analysis of fragmented ions – PFF
There are several different mass spectrometers. However, all sample molecules undergo the same processes regardless of instrument configuration. Sample molecules are introduced into the instrument through a sample inlet. Once inside the instrument, the sample molecules are converted to ions in the ionization source, before being electrostatically propelled into the mass analyzer. Ions are then separated according to their m/z within the mass analyzer. The detector converts the ion energy into electrical signals, which are then transmitted to a computer.
Ionization method refers to the mechanism of ionization while the ionization source is the mechanical device that allows ionization to occur. Ionization methods work by either ionizing a neutral molecule through electron ejection, electron capture, protonation, cationization, or deprotonation.
A proton is added to a molecule, producing a net positive charge of 1+ for every proton added. Positive charges tend to reside on the more basic residues of the molecule, such as amines, to form stable cations. Protonation can be achieved by MALDI and ESI.
The negative charge of 1- is achieved through the removal of a proton from a molecule. Deprotonation is more useful with acidic residues. Deprotonation can be achieved by MALDI or ESI.
Cationization involves adding positively charged ions by addition of a cation adduct such as alkali or ammonium. It is very suitable carbohydrates. Cationization can be achieved by MALDI or ESI.
This is generally achieved through the desorption or ejection of charged species from the condensed phase into the gas phase. This transfer is commonly achieved via MALDI or ESI.
MALDI and ESI are now the most common ionization sources for biomolecular mass spectrometry, offering excellent mass range and sensitivity.
| Ionization Source | Ionization Event |
|---|---|
| ESI | Evaporation of charged droplets |
| nanoESI | Evaporation of charged droplets |
| MALDI | Photon absorption/proton transfer |
The most important considerations for both MALDI and ESI are the physical state of the analyte and the ionization energy. Both instruments can produce positive or negative ions.
A good vacuum is needed to allow ions to reach the detector without undesirable collisions. Unwanted collisions would result in reduced resolution and sensitivity. A vulnerable spot is the point of sample insertion. ESI uses capillary column to maintain vacuum. MALDI evacuates the sample chamber with a vacuum lock.
| ESI | MALDI |
|---|---|
| Flow technique (LC,CE) | Pulse technique |
| Not very tolerant to salts (better off-axis) | More tolerant to impurities (wash) |
| Multiple charging: complex but useful | Generally singly charged |
| 1 fmol/ul possible with NanoSpray | Can consume less sample |
| Very high dynamic range | Lower dynamic range |
Actual results depend on sample, impurities and mass analyzer. Avoid or minimize salts, chaotropes, detergents, polymers, and non volatile compounds.
The analyte is mixed with a large excess of an aromatic ‘matrix compound’ that can absorb energy from the laser. The analyte and matrix are dissolved in an organic solvent and placed on a metallic probe or multiple-sample target. MALDI causes the ionization and transfer of a sample from the condensed phase to the gas phase via laser excitation of the sample. The solvent evaporates leaving matrix crystals in which the analyte is embedded.
Sample-matrix preparation procedures greatly influence the quality of MALDI mass spectra of peptides/proteins. Among the variety of reported preparation methods, the dried-droplet method is the most frequently used. MALDI is generally used with singly-charged ions with a high mass range analyzer such as TOF. MALDI can be used for high throughput microarrays on silicon chips, imaging of tissue or selection of individual cells or microorganisms.
MALDI requires samples to be dissolved in a matrix for analysis. Proteins are least soluble at their PI and therefore precipitate.
Detergents and salts should be reduced or removed from samples. Never use proteases and enzymes such as Cyanogen Bromide or trypsin in matrix preparation. They divide proteins.
Reference: http://www.chemistry.wustl.edu/~msf/damon/samp_prep_dried_droplet.html
In this method, the analyte and matrix solution are mixed together then loaded onto the MALDI sample plate. Solvents are dried by air drying. The disadvantage of this technique is the lack of signal reproducibility due to variability in drying conditions.
Mix analyte with a specially prepared matrix. This produces small crystal with a very uniform surface, improving mass accuracy.
It involves a fast solvent evaporation step to form the first layer of small crystals, followed by deposition of a mixture and analyte solution on top of the crystal layer. This technique keeps the benefits of fast evaporation and reduces its disadvantages. Therefore it is more accurate and sensitive.
When nitrocellulose is mixed with matrix solution, peptide ionization and signal reproducibility increases.
1. Matrix absorbs UV or IR energy from pulsed laser
2. Matrix ionizes and dissociates
3. Ions released by expanding plume
• Different lasers work with different matrices
• Absorb energy from laser wavelength
• Most matrices are acidic by nature
• Matrix can be hot (likely to fragment peptides) or cold (not likely to fragment peptides)
• Require high matrix-analyte ratio
• Several crystallization methods exist
Cold --> DHB --> larger molecules and proteins --> creates fewer fragments
Hot --> CHCA (alpha-cyano) --> better with peptides --> creates more fragments
Delayed extraction is a technique which allows ions to be extracted from ionization source after a cooling period of ~150 nanoseconds. This narrows the kinetic energy distribution of the ions, thus providing higher resolution than in continuous extraction techniques.
In delayed extraction mode, potential gradient does not exist when sample in ionized. Accelerating voltage is pulsed after a user-set time delay and ions are accelerated.
In continuous extraction mode, accelerating voltage is continuously applied, and the potential gradient exists when sample is ionized. Ions are accelerated immediately.
Delayed extraction improves mass resolution by reducing the spread in arrival times.
MALDI is widely used as a tool for peptides, proteins, and most other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids). The utility of heterogeneous samples makes it very attractive for mass analysis of complex biological samples such as proteolytic digests. MALDI is predominantly used for the analysis of simple peptide mixtures, such as the peptides derived from a single spot from a 2D-gel. The utility of MALDI for biomolecule analysis lies in its ability to provide molecular weight information on intact molecules.
| Advantages | Disadvantages |
|---|---|
| Practical mass range up to 300 000 Da | Background interference by matrix material |
| Good sensitivity - femtomole | Possibility of photo-degradation by laser |
| Soft ionization method with little or no fragmentation | Acidic matrix may degrade some compounds |
| Very tolerant to salts | |
| Suitable for analysis of complex mixtures | |
Soft ionization: not tearing the analyte apart. Soft ionization is capable of maintaining macromolcular complexes during ionization.
Most charged or ionisable molecules interfere with the ionization of the analyte (i.e. compete for charges) and cause signal suppression and/or elevation of the background noise. These molecules include salts, chaotrophes, detergents, polymers, and all non volatile and ionic compounds.
Good crystallization is essential for good results. If salt or detergent impedes crystallization, you might get no signal.
The analyte is dissolved and forced through a narrow needle held at high voltage. A fine spray of charged droplets emerges from the needle. These droplets are attracted to the entrance of the mass spectrometer due to high opposite voltage at the mass analyzer’s entrance. As they enter the mass spectrometer, the droplets are dried using a stream of inert gas, resulting is gas-phase ions that are accelerated through the analyzer towards the detector.
ESI is conducive to the formation of singly charged small molecules, but can also produce multiply charge species of larger molecules. Multiple charging makes it possible to observe very large molecules.
Many solvents can be used in ESI and are chosen based on the solubility of the compound of interest, the volatility of the solvent and the solvent’s ability to donate a proton. Better sensitivity is obtained when a volatile organic solvent is added.
ESI is generally used with multiply charged ions with quadrupoles and quadrupole ion traps. These instruments are more readily configured as tandem mass spectrometers for mass-selecting and fragmenting single components of a mixture.
Liquid technique is compatible with online chromatographic methods such as RP-HPLC, anion exchange chromatography and capillary electrophoresis.
ESI is a method routinely used with peptides, proteins, carbohydrates, small oligonucleotides, synthetic polymers, and lipids.
| Advantages | Disadvantages |
|---|---|
| Practical mass range up to 70000 Da | Salts and ion-pairing agents reduce sensibility |
| Good sensitivity - femtomole | Complex mixture can reduce sensitivity |
| Softest ionization method | Simultaneous mixture analysis can be poor |
| Easily adaptable to LC | Multiple charging can be confusing |
| Easily adaptable to tandem mass analyzers such as ion traps and triple quadrupoles | Sample purity is important |
| Multiple charging analysis allows high mass ion analysis | Carryover from sample to sample |
| No matrix interference |
nanoESI has a very small needle positioned close to the entrance of the mass analyzer, resulting in more efficient ion transmission. Effusing the sample at very low rates allows for high sensitivity. The end result of this rather simple adjustment is increased efficiency, which includes a reduction in the amount of sample needed.
Since nanoESI droplets are smaller, the amount of evaporation necessary to obtain ion formation is much less. As a consequence, nanoESI is more tolerant to salts and other impurities. Less evaporation means that impurities are not concentrated down as much as they are in ESI.
Analytical instruments in general have variations in their capabilities as a result of their individual design and intended purpose. Mass analyzers also have their variations their strengths and weaknesses associated with each variation. A mass analyzer measures gas phase ions with respect to their (m/z). It is important to remember that mass analyzers measure m/z ratio, not mass. Quadrupoles and TOFs separate ions in space. Ion trap separates ions in time.
| Mass Analyzers | Detection Method |
|---|---|
| Quadrupole | Scan radio frequency field (RF) |
| Quadrupole ion trap | Scan radio frequency field (RF) |
| TOF | TOF correlated directly to ion’s m/z |
| TOF Reflectron | TOF correlated directly to ion’s m/z |
| Quad-TOF | RF scanning + TOF |
| FT-ICR | Translates ion cyclotron motion to m/z |
Performance characteristics: The performance of a mass analyzer is judged by accuracy, resolution, mass range, tandem analysis capabilities, and scan speed.
Accuracy: The ability with which the analyzer can accurately provide m/z information. This is largely a function of an instrument’s stability and resolution.
Resolution: The ability of a mass spectrometer to distinguish between ions of different m/z ratios. So, greater resolution means increased ability to differentiate ions. As the data acquisition rate decreases, the resolution decreases as well.
Mass Range: The m/z range of the mass analyzer.
Tandem Mass Analysis: The ability of the analyzer to separate different molecular ions, generate fragment ions from a selected ion, and then measure the mass of fragmented ions. Fragmented ions are used for structural determination.
Scan speed: This refers to the rate at which the analyzer scans over a particular mass range.
Sensitivity: Sensitivity is an absolute quantity; resolution is a relative quantity. Sensitivity describes the smallest absolute amount of change that can be detected by a measurement. Sensitivity should not be confused with accuracy—they are entirely different parameters.
Purity: How well you can separate complex mixtures.
Cleanliness: Eliminate molecules that interfere with ionization/detection.
Isotopes: Isotopes are forms of an element whose nuclei have the same atomic number - the number of protons in the nucleus - but different mass numbers because they contain different numbers of neutrons. Isotopes have the same charge but different mass.
Tandem is time refers to precursor selection, dissociation, and fragment separation taking place in the same space but at different times. e.g. ion trap, FT-ICR.
The quadrupole is the most widely used analyzer due to its ease of use, mass range covered, good linearity for quantitative work, resolution and quality of mass spectra. Reasonably priced.
The main characteristics are:
A quadrupole can be operated in RF-only mode, which allows ions of any m/z ratio to pass through, or in scanning mode, where a potential difference is applied and the instrument acts as a mass filter.
A triple quadrupole has three quadrupoles arranged in a series. It can be set either for the analysis of intact peptides or their fragment ions.
Quadrupole separates ions in space.
Quadrupoles offer 3 main advantages:
U is voltage, V is RF. Mass spectrum is generated by increasing U and V at a constant ratio.
The mass window for observing an ion in SIM mode can be adjusted, in order to compensate small mass calibration shift. This is the span factor.
In Scan mode, the amplitude of the DC and RF voltages are ramped (while keeping a constant RF/DC ratio), to obtain the mass spectrum over the required mass range. The sensitivity is a function of the scanned mass range, scan speed, and resolution.
(1) Product ion scan. The precursor ion is focused in Q1 and transferred into Q2 - the collision cell - where it interacts with a collision gas and fragments. The fragments are then measured by scanning Q3. This results in the typical MS/MS spectrum and is the method most commonly employed with ESI ionization and/or LC-MS. Q1 fixed, Q3 scan. Gives structural information.
(2) Precursor ion scan. Q3 is held to measure the occurrence of a particular fragment ion and Q1 is scanned. This results in a spectrum of precursor ions that result in that particular product ion. Goal is to find all occurrence of a certain ion. Especially useful for EI and CI ionization. Q1 scan, Q3 fixed. Structural information & screening for analogues.
(3) Neutral loss scan. Q1 is scanned as in (2) but this time Q3 is also scanned to produce a spectrum of precursor ions that undergo a particular neutral loss. Again this mode is especially useful for EI and CI ionization. Q1 scan, Q3 scan – neutral offset. Structural information & screening for conjugates.
(4) Selected Reaction Monitoring. Q1 and Q3 are set to fixed masses. Goal is to detect a specific reaction. Which peptide would fragment into which fragments. –Both Q1 & Q2 are fixed. Target analysis & highest sensitivity.
TOF - Time of flight mass analyzers are the simplest mass analyzers. TOF analysis is based on accelerating a group of ions to a detector where all of the ions are given the same amount of energy through an accelerating potential. Given the same push, lighter ions reach the detector before the heavier ones. Mass, charge, and kinetic energy of the ions affect the arrival time and the detector.
Unlike quadrupole instruments, electric field is not required to separate ions. MALDI-TOF/TOF or hybrid analyzers are extremely sensitive. TOF separates ions in space.
Prompt Fragmentation is fragmentation before the push through the TOF.
Both ESI and MALDI can be used with ion trap analyzers. Ion trap consists of a chamber surrounded by a ring electrode and two end-cap electrodes. It can trap ions in a radio frequency quadrupole field.
Ions above a certain m/z threshold remain in the trap. Ions are ejected based on applied voltage. So a mass spectrum can be obtained by gradually increasing the voltage. Alternatively, an inert gas can be inserted to fragment the ions. Multiple rounds of fragmentation can be used.
Ions trap is capable of isolating ion species by ejecting all other from the trap. This is usually done to repeatedly fragment ions of interest. This significantly increases the amount of structural information which can be gathered.
Ion traps separate ions in time.
Ion trap is ideal for glycosylation analysis since it can break down sugars sequentially.
FT-ICR mass analyzer is the most complex and difficult to operate. It offers the highest resolution, mass accuracy, and sensitivity.
FTMS is based on the principle of monitoring a charged particle’s orbiting motion in a magnetic field. While ions are orbiting, a pulsed RF signal is used to excite them. This allows the ions to produce detectable image current by bringing them into coherent motion and enlarging the radius of the orbit. The image current can generated by the ions can then be Fourier-transformed to obtain component frequencies of different ions, which correspond to their m/z. All ions with the same m/z value will orbit with the same cyclotron frequency in a uniform magnetic field. Since frequencies can be obtained at high accuracy, m/z can also be determined at high accuracy. In addition to high resolution, FTMS offers the ability to perform MSn. It is capable of ejecting all but the ions of interest.
Unlike double sector instruments, FT-ICR does not suffer from loss of sensitivity at high resolutions.
A hybrid mass analyzer is a mixture of two or more mass analyzers. If done correctly, a hybrid can couple the benefits of different mass analyzers.
qTOF combines the stability of a quadrupole analyzer with the high efficiency, sensitivity, and accuracy of a TOF reflectron mass analyzer. The quadrupole can act as any simple quadrupole analyzer to scan across a specified m/z range. However, it can also be used to selectively isolate a precursor ion and direct that ion into the collision cell. The resultant fragment ions are then analyzed by the TOF reflectron mass analyzer.
qTOF exploits the quadrupole’s ability to select a particular ion and the ability of TOF to achieve simultaneous and accurate measurements of ions across full mass range. qTOF offers significantly higher sensitivity and accuracy over tandem quadrupole instruments when acquiring full fragment mass spectra.
Once separated by mass analyzer, ions reach ion detector which generates a current signal from incident ions. The most commonly used detector is the electron multiplier.3 different types of detectors: Electron multipliers, dynolyte photomultiplier, microchannel plates.
A conversion dynode is used to convert either negative or positive ions into electrons. These electrons are amplified by a cascade effect in a horn shape device, to produce a current. This device, also called channeltron, is widely used in quadrupole and ion trap instruments.
Ions exiting the quadrupole are converted to electrons by a conversion dynode.These electrons strike a phosphor which when excited, emit photons. The photons strike a photocathode at the front of the photomultiplier to produce electrons and the signal is amplified by the photomultiplier. The photomultiplier is sealed in glass and held under vacuum. This prevents contamination and allows the detector to maintain its performance for a considerably longer period than conventional electron multipliers.
Most TOF spectrometers employ multichannel plate (mcp) detectors which have a time response < 1 ns and a high sensitivity (single ion signal > 50 mV). The large and plane detection area of mcp's results in a large acceptance volume of the spectrometer system. Only few mcp channels out of thousands are affected by the detection of a single ion i.e. it is possible to detect many ions at the same time which is important for laser ionisation where hundreds of ions can be created within a few nanoseconds. dynode phosphor photomultiplier.
PMF is an analytical technique for protein identification using data from intact peptide masses. A protease such as Trypsin is used to cleave a protein of interest. The masses of the proteins are measured with a mass spectrometer. Each protein can be uniquely identified by the masses of its constituent peptides since protein masses are extremely discriminatory. The Accuracy of PMF depends on quality and relative intensities of the peaks, mass accuracy of the instrument, and interfering factors such are PTMs. PMF can only be used to identify proteins which are sequenced. Therefore PMF is best suited to those organisms whose cDNA protein sequence data is available in a database. It must be noted that even small differences in mass can result in faulty results since PMF accuracy depends entirely on the accurate correlation of determined and predicted masses.
PMF relies on proteases to digest the protein into smaller peptides. Different proteases cut the proteins at different amino acids. An enzyme of low specificity, which digests a protein into too many peptides or results in many missed cleavages, should not be used. A complex mixture result is overlapping peaks. A missed cleavage is when a protease doesn’t cleave where it is supposed to. Trypsin or an enzyme with similar or higher specificity is a good choice. One should expect 1 or 2 missed cleavages from Trypsin per protein. The mass of a peptide, regardless of the protease used to cleave it, is the sum of the amino acids present in the peptide. The effects of modification which might be made on the amino acids must also be taken into account. For example, a phosphorylation results in an addition of a phosphate group, an addition of about 80 Daltons.
Mascot is tool which uses protein mass spectrometry data to identify proteins from primary sequence databases. This tool allows peptide mass fingerprinting. It allows you to specify the enzyme used, missed cleavages expected.
When PMF fails, fragments in the CID spectrum can provide crucial information. The data can be used in two ways:
In order to obtain peptide sequence information by mass spectrometry, fragments of an ion must be produced that reflect structural features of the original compound. Fortunately, most peptides are linear molecules, which allow for relatively straightforward interpretation of the fragmentation data. This is accomplished by colliding the ions with an inert gas. The fragments then monitored via mass analysis.
Tandem mass spectrometry allows for a heterogeneous solution of peptides to be analyzed by filtering the ion of interest into the collision cell, structural information can be derived on each peptide from a complex mixture. The fragment ions produced in this process can be separated into two classes. Once class retains the charge on the N-terminus and fragmentation occurs at a, b, and c. The second class of fragment ions retain the charge on the C-terminus and fragmentation occurs at x, y, and z. Most fragments are obtained from cleavage between a carbonyl and amide bond.
In determining the amino acid sequence of a peptide, it is not possible to distinguish between L and I because they have the same mass.
Since a complete ion series (y or b) is not observed, the combination of the two series can provide useful information for protein identification.
In multiply-charged peptides, the proton is strongly delocalized. This results in rich fragmentation and good sequence coverage. In singly-charged peptides, the proton is localized on basic residues. This results in poor fragmentation and low sequence coverage.
Important considerations for obtaining high quality signal are sample solubility, matrix selection, ionization characteristics, salt content and purity. Factors affecting data interpretation include quantitation, molecular weight calculation, isotope patterns, calibration/accuracy, sensitivity, and speed.
Ability of a molecule to become ionized is closely related to its functional groups. The best quantitation is obtained when a compound is calibrated against an internal standard similar to the molecule in question.
The resolving power of a mass spectrometer is very important in calculating mass of a molecule.
It is important to distinguish between internal and external calibration. External calibration refers to the instrument being calibrated followed by analysis without the present of a calibrant. Internal calibration refers to analyses that are performed with a calibrant present to improve accuracy.
Mass spectrometry can be used to determine both primary and higher order structures of proteins. The basis for these investigations lies in the ability of mass analysis techniques to detect changes in protein conformation under differing conditions. These experiments include:
The accuracy and sensitivity of PMF allows for the exploration of protein structure and even structural dynamics.
PMF combines enzymatic digestion, mass spectrometry, and sequence specific data analysis to produce and examine proteolytic fragments. This information could then be used to identify protein and get information about protein structure.
Higher order structure of a protein can be evaluated when PMF techniques are combined with limited proteolytic digestion. Limited proteolysis refers to the exposure of a protein or complex to digestion conditions that last for a brief period. This is performed to gain information on the parts of the protein exposed to the surface.
The sequence specificity of the proteolytic enzyme plays a major role in the application of mass spectrometry to protein structure. A sequence specific protease reduces the number of fragments that are produced and, concomitantly, improves the likelihood for statistically matches. Accessibility and flexibility of a protein is also very important. Surface proteins are usually hydrophilic so proteases that cleave hydrophilic sites are preferred.
PMF can be used to recognize simple conformational differences between protein states. After conformational changes, the same protein would digest into different mass maps.
ESI has been used to monitor protein folding and protein complexes. Some proteins exhibit a distinct difference in their charge state distribution which is a reflection of their solution conformation. ESI is a simple but highly sensitive and informative method to characterize the functional shape(s) of proteins (globular or extended) prior to more material-intensive and time-consuming spectroscopic or crystallographic studies.
Absolute quantitation: determine the absolute concentration of proteins/peptides in a selected fluid/cell/tissue: can apply to a series of samples.
Relative quantitation: determine the concentration of proteins/peptides by comparison to an internal standard and/or similar fluid/cell/tissue at a different physiological stage.
ICAT uses stable isotope labeling to perform quantitative analysis of paired protein samples, followed by separation and identification of proteins within these complex mixtures by LC-MS. The isotopic tags bind covalently to Cys within a protein. The tags are almost identical, possessing the same structure and chemical properties, but exist in two isotopic forms:
• Light - possessing eight hydrogens
• Heavy - possessing eight deutriums
When bound to the same peptide, a concrete mass change of exactly 8 mass units will be evident when analyzed by MS.
The tag has three functional elements:
1. a biotin tag, used during affinity capture
2. isotopically encoded linker chain
3. reactive group which will bind to and modify Cys residues
Using ICAT is a 4 step process:
1. free cysteines in a protein are reacted with a special affinity tag
2. labeled proteins are enzymatically digested
3. labeled peptides are separated from bulk using LC prior to MS
4. MS detects the mass differences in the same peptides
Two samples are separately treated with affinity tags. One with light ICAT and the other with heavy ICAT. The samples are mixed, digested and passed through MS.
The strength of this technique lies in its ability to allow quantification and identification within a single analysis. It also can be applied to samples from any source as it does not require metabolic labeling. The advantage over 2D gel is its speed and ease of automation.
Weaknesses of this method include the frequent need for extensive sample fractionation before MS/MS analysis. Since the procedure targets Cys residues, proteins that do not contain Cys cannot be quantified. This represents about 10% of the proteins.
- MPB slides
- Expanding role of mass spectrometry in biotechnology by Gary Siuzdak
- Principles of proteomics by R. M. Twyman
- Wikipedia.org
- http://www.waters.com/watersdivision/ContentD.asp?watersit=EGOO-66MNYR