An Introduction to Mass Spectrometry P2

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6. Matrix assisted laser desorption ionisation
Matrix Assisted Laser Desorption Ionisation (MALDI) (F. Hillenkamp, M. Karas, R. C. Beavis, B. T. Chait, Anal. Chem., 1991, 63, 1193) deals well with thermolabile, non-volatile organic compounds especially those of high molecular mass and is used successfully in biochemical areas for the analysis of proteins, peptides, glycoproteins, oligosaccharides, and oligonucleotides. It is relatively straightforward to use and reasonably tolerant to buffers and other additives. The mass accuracy depends on the type and performance of the analyser of the mass spectrometer, but most modern instruments should be capable of measuring masses to within 0.01% of the molecular mass of the sample, at least up to ca. 40,000 Da.
MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionisation. The sample is pre-mixed with a highly absorbing matrix compound for the most consistent and reliable results, and a low concentration of sample to matrix works best. The matrix transforms the laser energy into excitation energy for the sample, which leads to sputtering of analyte and matrix ions from the surface of the mixture. In this way energy transfer is efficient and also the analyte molecules are spared excessive direct energy that may otherwise cause decomposition. Most commercially available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength 337 nm.

Matrix assisted laser desorption ionisation (MALDI)

The sample to be analysed is dissolved in an appropriate volatile solvent, usually with a trace of trifluoroacetic acid if positive ionisation is being used, at a concentration of ca. 10 pmol/�L and an aliquot (1-2 �L) of this removed and mixed with an equal volume of a solution containing a vast excess of a matrix. A range of compounds is suitable for use as matrices: sinapinic acid is a common one for protein analysis while alpha-cyano-4-hydroxycinnamic acid is often used for peptide analysis. An aliquot (1-2 �L) of the final solution is applied to the sample target which is allowed to dry prior to insertion into the high vacuum of the mass spectrometer. The laser is fired, the energy arriving at the sample/matrix surface optimised, and data accumulated until a m/z spectrum of reasonable intensity has been amassed. The time-of-flight analyser separates ions according to their mass(m)-to-charge(z) (m/z) ratios by measuring the time it takes for ions to travel through a field free region known as the flight, or drift, tube. The heavier ions are slower than the lighter ones.
The m/z scale of the mass spectrometer is calibrated with a known sample that can either be analysed independently (external calibration) or pre-mixed with the sample and matrix (internal calibration).

Simplified schematic of MALDI-TOF mass spectrometry (linear mode)

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.
In positive ionisation mode the protonated molecular ions (M+H⁺) are usually the dominant species, 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. Positive ionisation is used in general for protein and peptide analyses.
In negative ionisation mode the deprotonated molecular ions (M-H^-) are usually the most abundant species, accompanied by some salt adducts and possibly traces of dimeric or doubly charged materials. Negative ionisation can be used for the analysis of oligonucleotides and oligosaccharides.

Positive ionisation MALDI m/z spectrum of a peptide mixture using alpha-cyano-4-hydroxycinnamic acid as matrix

7. Positive or negative ionisation?
If the sample has functional groups that readily accept a proton (H⁺) then positive ion detection is used
e.g. amines R-NH₂ + H⁺ = R-NH₃⁺ as in proteins or peptides.
If the sample has functional groups that readily lose a proton then negative ion detection is used
e.g. carboxylic acids R-CO₂H = R-CO₂^- and alcohols R-OH = R-O- as in saccharides or oligonucleotides
8. Tandem mass spectrometry (MS-MS): Structural and sequence information from mass spectrometry.
8.1 Tandem mass spectrometry
Tandem mass spectrometry (MS-MS) is used to produce structural information about a compound by fragmenting specific sample ions inside the mass spectrometer and identifying the resulting fragment ions. This information can then be pieced together to generate structural information regarding the intact molecule. Tandem mass spectrometry also enables specific compounds to be detected in complex mixtures on account of their specific and characteristic fragmentation patterns.
A tandem mass spectrometer is a mass spectrometer that has more than one analyser, in practice usually two. The two analysers are separated by a collision cell into which an inert gas (e.g. argon, xenon) is admitted to collide with the selected sample ions and bring about their fragmentation. The analysers can be of the same or of different types, the most common combinations being:

quadrupole - quadrupole
magnetic sector - quadrupole
magnetic sector - magnetic sector
quadrupole - time-of-flight.

Fragmentation experiments can also be performed on certain single analyser mass spectrometers such as ion trap and time-of-flight instruments, the latter type using a post-source decay experiment to effect the fragmentation of sample ions.
8.2 Tandem mass spectrometry analyses.
The basic modes of data acquisition for tandem mass spectrometry experiments are as follows:
Product or daughter ion scanning:
the first analyser is used to select user-specified sample ions arising from a particular component; usually the molecular-related (i.e. (M+H)⁺ or (M-H)^-) ions. These chosen ions pass into the collision cell, are bombarded by the gas molecules which cause fragment ions to be formed, and these fragment ions are analysed i.e. separated according to their mass to charge ratios, by the second analyser. All the fragment ions arise directly from the precursor ions specified in the experiment, and thus produce a fingerprint pattern specific to the compound under investigation.
This type of experiment is particularly useful for providing structural information concerning small organic molecules and for generating peptide sequence information.
Precursor or parent ion scanning:
the first analyser allows the transmission of all sample ions, whilst the second analyser is set to monitor specific fragment ions, which are generated by bombardment of the sample ions with the collision gas in the collision cell. This type of experiment is particularly useful for monitoring groups of compounds contained within a mixture which fragment to produce common fragment ions, e.g. glycosylated peptides in a tryptic digest mixture, aliphatic hydrocarbons in an oil sample, or glucuronide conjugates in urine.
Constant neutral loss scanning:
this involves both analysers scanning, or collecting data, across the whole m/z range, but the two are off-set so that the second analyser allows only those ions which differ by a certain number of mass units (equivalent to a neutral fragment) from the ions transmitted through the first analyser. e.g. This type of experiment could be used to monitor all of the carboxylic acids in a mixture. Carboxylic acids tend to fragment by losing a (neutral) molecule of carbon dioxide, CO₂, which is equivalent to a loss of 44 Da or atomic mass units. All ions pass through the first analyser into the collision cell. The ions detected from the collision cell are those from which 44 Da have been lost.
Selected/multiple reaction monitoring:
both of the analysers are static in this case as user-selected specific ions are transmitted through the first analyser and user-selected specific fragments arising from these ions are measured by the second analyser. The compound under scrutiny must be known and have been well-characterised previously before this type of experiment is undertaken. This methodology is used to confirm unambiguously the presence of a compound in a matrix e.g. drug testing with blood or urine samples. It is not only a highly specific method but also has very high sensitivity.
8.3 Peptide Sequencing by Tandem Mass Spectrometry.
The most common usage of MS-MS in biochemical areas is the product or daughter ion scanning experiment which is particularly successful for peptide and nucleotide sequencing.
Peptide sequencing: H₂N-CH(R')-CO-NH-CH(R")-CO₂H
Peptides fragment in a reasonably well-documented manner (P. Roepstorrf, J. Fohlmann, Biomed. Mass Spectrom., 1984, 11, 601; R. S. Johnson, K. Biemann, Biomed. Environ. Mass Spectrom., 1989, 18, 945). The protonated molecules fragment along the peptide backbone and also show some side-chain fragmentation with certain instruments (Four-Sector Tandem Mass Spectrometry of Peptides, A. E. Ashcroft, P. J. Derrick in "Mass Spectrometry of Peptides" ed. D. M. Desiderio, CRC Press, Florida, 1990).
There are three different types of bonds that can fragment along the amino acid backbone: the NH-CH, CH-CO, and CO-NH bonds. Each bond breakage gives rise to two species, one neutral and the other one charged, and only the charged species is monitored by the mass spectrometer. The charge can stay on either of the two fragments depending on the chemistry and relative proton affinity of the two species. Hence there are six possible fragment ions for each amino acid residue and these are labelled as in the diagram, with the a, b, and c" ions having the charge retained on the N-terminal fragment, and the x, y", and z ions having the charge retained on the C-terminal fragment. The most common cleavage sites are at the CO-NH bonds which give rise to the b and/or the y" ions. The mass difference between two adjacent b ions, or y"; ions, is indicative of a particular amino acid residue (see Table of amino acid residues at the end of this document).

Peptide sequencing by tandem mass spectrometry - backbone cleavages

The extent of side-chain fragmentation detected depends on the type of analysers used in the mass spectrometer. A magnetic sector - magnetic sector instrument will give rise to high energy collisions resulting in many different types of side-chain cleavages. Quadrupole - quadrupole and quadrupole - time-of-flight mass spectrometers generate low energy fragmentations with fewer types of side-chain fragmentations.
Immonium ions (labelled "i") appear in the very low m/z range of the MS-MS spectrum. Each amino acid residue leads to a diagnostic immonium ion, with the exception of the two pairs leucine (L) and iso-leucine (I), and lysine (K) and glutamine (Q), which produce immonium ions with the same m/z ratio, i.e. m/z 86 for I and L, m/z 101 for K and Q. The immonium ions are useful for detecting and confirming many of the amino acid residues in a peptide, although no information regarding the position of these amino acid residues in the peptide sequence can be ascertained from the immonium ions.
An example of an MS/MS daughter or product ion spectrum is illustrated below. The molecular mass of the peptide was measured using standard mass spectrometric techniques and found to be 680.4 Da, the dominant ions in the MS spectrum being the protonated molecular ions (M+H⁺) at m/z 681.4. These ions were selected for transmission through the first analyser, then fragmented in the collision cell and their fragments analysed by the second analyser to produce the following MS/MS spectrum. The sequence (amino acid backbone) ions have been identified, and in this example the peptide fragmented predominantly at the CO-NH bonds and gave both b and y" ions. (Often either the b series or the y" series predominates, sometimes to the exclusion of the other). The b series ions have been labelled with blue vertical lines and the y" series ions have been labelled with red vertical lines. The mass difference between adjacent members of a series can be calculated e.g. b3-b2 = 391.21 - 262.16 = 129.05 Da which is equivalent to a glutamine (E) amino acid residue; and similarly y4 - y3 = 567.37 - 420.27 = 147.10 Da which is equivalent to a phenylalanine (F) residue. In this way, using either the b series or the y" series, the amino acid sequence of the peptide can be determined and was found to be NFESGK (n.b. the y" series reads from right to left!). The immonium ions at m/z 102 merely confirm the presence of the glutamine (E) residue in the peptide.

Peptide sequencing by tandem mass spectrometry - an MS-MS daughter or product ion spectrum.

A protein identification study would proceed as follows:

a. The protein under investigation would be analysed by mass spectrometry to generate a molecular mass to within an accuracy of 0.01%.
b. The protein would then be digested with a suitable enzyme. Trypsin is useful for mass spectrometric studies because each proteolytic fragment contains a basic arginine (R) or lysine (K) amino acid residue, and thus is eminently suitable for positive ionisation mass spectrometric analysis. The digest mixture is analysed - without prior separation or clean-up - by mass spectrometry to produce a rather complex spectrum from which the molecular weights of all of the proteolytic fragments can be read. This spectrum, with its molecular weight information, is called a peptide map. (If the protein already exists on a database, then the peptide map is often sufficient to confirm the protein.)
For these experiments the mass spectrometer would be operated in the "MS" mode, whereby the sample is sprayed and ionised from the nanospray needle and the ions pass through the sampling cone, skimmer lenses, Rf hexapole focusing system, and the first (quadrupole) analyser. The quadrupole in this instance is not used as an analyser, merely as a lens to focus the ion beam into the second (time-of-flight) analyser which separates the ions according to their mass-to-charge ratio.

Q-TOF mass spectrometer operating in MS (upper) and MS/MS mode (lower) modes.

c. With the digest mixture still spraying into the mass spectrometer, the Q-Tof mass spectrometer is switched into "MS/MS" mode. The protonated molecular ions of each of the digest fragments can be independently selected and transmitted through the quadrupole analyser, which is now used as an analyser to transmit solely the ions of interest into the collision cell which lies inbetween the first and second analysers. An inert gas such as argon is introduced into the collision cell and the sample ions are bombarded by the collision gas molecules which cause them to fragment. The optimum collision cell conditions vary from peptide to peptide and must be optimised for each one. The fragment (or daughter or product) ions are then analysed by the second (time-of-flight) analyser. In this way an MS/MS spectrum is produced showing all the fragment ions that arise directly from the chosen parent or precursor ions for a given peptide component.

An MS/MS daughter (or fragment, or product) ion spectrum is produced for each of the components identified in the proteolytic digest. Varying amounts of sequence information can be gleaned from each fragmentation spectrum, and the spectra need to be interpreted carefully. Some of the processing can be automated, but in general the processing and interpretation of spectra will take longer than the data acquisition if accurate and reliable data are to be generated.

The amount of sequence information generated will vary from one peptide to another, Some peptide sequences will be confirmed totally, other may produce a partial sequence of, say, 4 or 5 amino acid residues. Often sequence "tag" of 4 or 5 residues is sufficient to search a protein database and confirm the identity of the protein.
Peptide sequencing in summary:
Peptides fragment along the amino acid backbone to give sequence information.
Peptides ca. 2500 Da or less produce the most useful data.
The amount of sequence information varies from one peptide to another. Some peptides can generate sufficient information for a full sequence to be determined; others may generate a partial sequence of 4 or 5 amino acids.
A protein digest can be analysed as an entire reaction mix, without any separation of the products, from which individual peptides are selected and analysed by the mass spectrometer to generate sequence information.
About 4 �L of solution is required for the analysis of the digest mixture, with a concentration based on the original protein of ca. 1-10 pmol/�L. MS/MS sequencing is a sensitive technique consuming little sample.
Sometimes the full protein sequence can be verified; some proteins generate sufficient information to cover only part of the sequence. 70 - 80% coverage is reasonable.
Often a sequence "tag" of 4/5 amino acids from a single proteolytic peptide is sufficient to identify the protein from a database.
The final point in this summary means that mass spectrometers have been found to be extremely useful for proteomic studies, as illustrated below.
The proteomics procedure usually involves excising individual spots from a 2-D gel and independently enzymatically digesting the protein(s) contained within each spot, before analysing the digest mixture by mass spectrometer in the manner outlined above. Electrospray ionisation or MALDI could be used at this step.
The initial MS spectrum determining the molecular masses of all of the components in the digest mixture can often provide sufficient information to search a database using just several of the molecular weights from this peptide map.
If the database search is not fruitful, either because the protein has not been catalogued, is previously uncharacterised, or the data are not accurate or comprehensive enough to distinguish between several entries in the database, then further information is required.
This can be achieved by sample clean-up and then MS/MS studies to determine the amino acid sequences of the individual proteolytic peptides contained in the digest mixture, with which further database searching can be carried out.
8.4 Oligonucleotide sequencing by Tandem Mass Spectrometry.
Oligonucleotide sequencing: P-S(B)-P-S(B)-P-S(B)
Oligonucleotide sequencing can also be achieved by tandem mass spectrometry although it is not so well documented. However fragmentation patterns have been established and reported (S. Pomerantz, J. A. Kowalak, J. A. McClosky, J. Amer. Soc. Mass Spectrom., 1993, 4, 204). The experimental principle is similar to that of peptide sequencing, in that individual species are mass measured in MS mode of instrument operation, and then their molecular-related ions selected by the first (quadrupole) analyser to be transmitted into the collision cell where they undergo fragmentation after bombardment with a collision gas. The fragments are analysed by the second (time-of-flight) analyser to produce an MS/MS product, or daughter, ion spectrum showing all the fragment ions that arise directly from the chosen parent or precursor ions.
Negative electrospray ionisation is often the preferred ionisation method. The optimisation of the fragmentation conditions varies from component to component and diligence must be taken to ensure the best conditions are employed.
Data processing and interpretation is again of paramount importance for accurate, reliable results and hence sequence information.
9. General reading
"Mass Spectrometry: A Foundation Course", K. Downard, Royal Society of Chemistry, UK, 2004.
"An Introduction to Biological Mass Spectrometry", C. Dass, Wiley, USA, 2002.
"The Expanding Role of Mass Spectrometry in Biotechnology", G. Siuzdak, MCC Press, San Diego, 2004.
"Ionization Methods in Organic Mass Spectrometry", A.E. Ashcroft, Analytical Monograph, Royal Society of Chemistry, UK, 1997.
[You must be registered and logged in to see this link.] (A.E. Ashcroft's MS web pages and tutorial)
Table of amino acid residues .

Symbol	Structure	Mass (Da)
Ala A	-NH.CH.(CH₃).CO-	71.0
Arg R	-NH.CH.[(CH₂)₃.NH.C(NH).NH₂].CO-	156.1
Asn N	-NH.CH.(CH₂CONH₂).CO-	114.0
Asp D	-NH.CH.(CH₂COOH).CO-	115.0
Cys C	-NH.CH.(CH₂SH).CO-	103.0
Gln Q	-NH.CH.(CH₂CH₂CONH₂).CO-	128.1
Glu E	-NH.CH.(CH₂CH₂COOH).CO-	129.0
Gly G	-NH.CH₂.CO-	57.0
His H	-NH.CH.(CH₂C₃H₃N₂).CO-	137.1
Ile I	-NH.CH.[CH.(CH₃)CH₂.CH₃].CO-	113.1
Leu	-NH.CH.[CH₂CH(CH₃)₂].CO-	113.1
Lys K	-NH.CH.[(CH₂)₄NH₂].CO-	128.1
Met M	-NH.CH.[(CH₂)₂.SCH₃].CO-	131.0
Phe F	-NH.CH.(CH₂Ph).CO-	147.1
Pro P	-NH.(CH₂)₃.CH.CO-	97.1
Ser S	-NH.CH.(CH₂OH).CO-	87.0
Thr T	-NH.CH.[CH(OH)CH₃).CO-	101.0
Trp W	-NH.CH.[CH₂.C₈H₆N].CO-	186.1
Tyr Y	-NH.CH.[(CH₂).C₆H₄.OH].CO-	163.1
Val V	-NH.CH.[CH(CH₃)₂].CO-	99.1

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