An Introduction to Mass Spectrometry P1

Tiêu đề: An Introduction to Mass Spectrometry P1 Fri Jan 14, 2011 2:50 pm

An Introduction to Mass Spectrometry

Dr Alison E. Ashcroft,
Mass Spectrometry Facility Manager,
Astbury Centre for Structural Molecular Biology,
Astbury Building ,
The University of Leeds.

CONTENTS

What is mass spectrometry (MS)? What Information does mass spectrometry provide?
Where are mass spectrometers used?
How can mass spectrometry help biochemists?
How does a mass spectrometer work?
1. Introduction
2. Sample introduction
3. Methods of sample ionisation
4. Analysis and separation of sample ions
5. Detection and recording of sample ions
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Electrospray ionisation
1. Electrospray ionisation
2. Nanospray ionisation
3. Data processing
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Matrix assisted laser desorption ionisation
Positive or negative ionisation?
Tandem mass spectrometry (MS-MS): Structural and sequence information from mass spectrometry
1. Tandem mass spectrometry
2. Tandem mass spectrometry analyses
3. Peptide sequencing by tandem mass spectrometry
4. Oligonucleotide sequencing by tandem mass spectrometry
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Background reading

1. What is mass spectrometry (MS)? What information does mass spectrometry provide?
Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample.
For large samples such as biomolecules, molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample i.e. within a 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected, e.g. the substitution of one amino acid for another, or a post-translational modification.
For small organic molecules the molecular mass can be measured to within an accuracy of 5 ppm or less, which is often sufficient to confirm the molecular formula of a compound, and is also a standard requirement for publication in a chemical journal.
Structural information can be generated using certain types of mass spectrometers, usually those with multiple analysers which are known as tandem mass spectrometers. This is achieved by fragmenting the sample inside the instrument and analysing the products generated. This procedure is useful for the structural elucidation of organic compounds and for peptide or oligonucleotide sequencing.
2. Where are mass spectrometers used?
Mass spectrometers are used in industry and academia for both routine and research purposes. The following list is just a brief summary of the major mass spectrometric applications:

Biotechnology: the analysis of proteins, peptides, oligonucleotides
Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism
Clinical: neonatal screening, haemoglobin analysis, drug testing
Environmental: PAHs, PCBs, water quality, food contamination
Geological: oil composition

3. How can mass spectrometry help biochemists?

Accurate molecular weight measurements:
sample confirmation, to determine the purity of a sample, to verify amino acid substitutions, to detect post-translational modifications, to calculate the number of disulphide bridges
Reaction monitoring:
to monitor enzyme reactions, chemical modification, protein digestion
Amino acid sequencing:
sequence confirmation, de novo characterisation of peptides, identification of proteins by database searching with a sequence "tag" from a proteolytic fragment
Oligonucleotide sequencing:
the characterisation or quality control of oligonucleotides
Protein structure:
protein folding monitored by H/D exchange, protein-ligand complex formation under physiological conditions, macromolecular structure determination

4. How does a mass spectrometer work?
4.1 Introduction
Mass spectrometers can be divided into three fundamental parts, namely the ionisation source , the analyser , and the detector.
The sample has to be introduced into the ionisation source of the instrument. Once inside the ionisation source, the sample molecules are ionised, because ions are easier to manipulate than neutral molecules. These ions are extracted into the analyser region of the mass spectrometer where they are separated according to their mass (m) -to-charge (z) ratios (m/z) . The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum .
The analyser and detector of the mass spectrometer, and often the ionisation source too, are maintained under high vacuum to give the ions a reasonable chance of travelling from one end of the instrument to the other without any hindrance from air molecules. The entire operation of the mass spectrometer, and often the sample introduction process also, is under complete data system control on modern mass spectrometers.
4.2 Sample introduction
The method of sample introduction to the ionisation source often depends on the ionisation method being used, as well as the type and complexity of the sample.
The sample can be inserted directly into the ionisation source, or can undergo some type of chromatography en route to the ionisation source. This latter method of sample introduction usually involves the mass spectrometer being coupled directly to a high pressure liquid chromatography (HPLC), gas chromatography (GC) or capillary electrophoresis (CE) separation column, and hence the sample is separated into a series of components which then enter the mass spectrometer sequentially for individual analysis.
4.3 Methods of sample ionisation

Many ionisation methods are available and each has its own advantages and disadvantages ("Ionization Methods in Organic Mass Spectrometry", Alison E. Ashcroft, The Royal Society of Chemistry, UK, 1997; and references cited therein).
The ionisation method to be used should depend on the type of sample under investigation and the mass spectrometer available.
Ionisation methods include the following:
Atmospheric Pressure Chemical Ionisation (APCI)
Chemical Ionisation (CI)
Electron Impact (EI)
Electrospray Ionisation (ESI)
Fast Atom Bombardment (FAB)
Field Desorption / Field Ionisation (FD/FI)
Matrix Assisted Laser Desorption Ionisation (MALDI)
Thermospray Ionisation (TSP)
The ionisation methods used for the majority of biochemical analyses are Electrospray Ionisation (ESI) and Matrix Assisted Laser Desorption Ionisation (MALDI) , and these are described in more detail in Sections 5 and 6 respectively.
With most ionisation methods there is the possibility of creating both positively and negatively charged sample ions, depending on the proton affinity of the sample. Before embarking on an analysis, the user must decide whether to detect the positively or negatively charged ions (see section 7).
4.4 Analysis and Separation of Sample Ions
The main function of the mass analyser is to separate , or resolve , the ions formed in the ionisation source of the mass spectrometer according to their mass-to-charge (m/z) ratios. There are a number of mass analysers currently available, the better known of which include quadrupoles , time-of-flight (TOF) analysers, magnetic sectors , and both Fourier transform and quadrupole ion traps .
These mass analysers have different features, including the m/z range that can be covered, the mass accuracy, and the achievable resolution. The compatibility of different analysers with different ionisation methods varies. For example, all of the analysers listed above can be used in conjunction with electrospray ionisation, whereas MALDI is not usually coupled to a quadrupole analyser.
Tandem (MS-MS) mass spectrometers are instruments that have more than one analyser and so can be used for structural and sequencing studies. Two, three and four analysers have all been incorporated into commercially available tandem instruments, and the analysers do not necessarily have to be of the same type, in which case the instrument is a hybrid one. More popular tandem mass spectrometers include those of the quadrupole-quadrupole, magnetic sector-quadrupole , and more recently, the quadrupole-time-of-flight geometries.
4.5 Detection and recording of sample ions.
The detector monitors the ion current, amplifies it and the signal is then transmitted to the data system where it is recorded in the form of mass spectra . The m/z values of the ions are plotted against their intensities to show the number of components in the sample, the molecular mass of each component, and the relative abundance of the various components in the sample.
The type of detector is supplied to suit the type of analyser; the more common ones are the photomultiplier , the electron multiplier and the micro-channel plate detectors. 5. Electrospray ionisation
5.1 Electrospray ionisation
Electrospray Ionisation (ESI) is one of the Atmospheric Pressure Ionisation (API) techniques and is well-suited to the analysis of polar molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular mass.

Standard electrospray ionisation source (Platform II)

During standard electrospray ionisation (J. Fenn, J. Phys. Chem., 1984, 88, 4451), the sample is dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary (75 - 150 micrometers i.d.) at a flow rate of between 1 �L/min and 1 mL/min. A high voltage of 3 or 4 kV is applied to the tip of the capillary, which is situated within the ionisation source of the mass spectrometer, and as a consequence of this strong electric field, the sample emerging from the tip is dispersed into an aerosol of highly charged droplets, a process that is aided by a co-axially introduced nebulising gas flowing around the outside of the capillary. This gas, usually nitrogen, helps to direct the spray emerging from the capillary tip towards the mass spectrometer. The charged droplets diminish in size by solvent evaporation, assisted by a warm flow of nitrogen known as the drying gas which passes across the front of the ionisation source. Eventually charged sample ions, free from solvent, are released from the droplets, some of which pass through a sampling cone or orifice into an intermediate vacuum region, and from there through a small aperture into the analyser of the mass spectrometer, which is held under high vacuum. The lens voltages are optimised individually for each sample.

The electrospray ionisation process

5.2 Nanospray ionisation
Nanospray ionisation (M. Wilm, M. Mann, Anal. Chem., 1996, 68, 1) is a low flow rate version of electrospray ionisation. A small volume (1-4 microL) of the sample dissolved in a suitable volatile solvent, at a concentration of ca. 1 - 10 pmol/microL, is transferred into a miniature sample vial. A reasonably high voltage (ca. 700 - 2000 V) is applied to the specially manufactured gold-plated vial resulting in sample ionisation and spraying. The flow rate of solute and solvent using this procedure is very low, 30 - 1000 nL/min, and so not only is far less sample consumed than with the standard electrospray ionisation technique, but also a small volume of sample lasts for several minutes, thus enabling multiple experiments to be performed. A common application of this technique is for a protein digest mixture to be analysed to generate a list of molecular masses for the components present, and then each component to be analysed further by tandem mass spectrometric (MS-MS) amino acid sequencing techniques (see Section . ESI and nanospray ionisation are very sensitive analytical techniques but the sensitivity deteriorates with the presence of non-volatile buffers and other additives, which should be avoided as far as possible.

In positive ionisation mode, a trace of formic acid is often added to aid protonation of the sample molecules; in negative ionisation mode a trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules. Proteins and peptides are usually analysed under positive ionisation conditions and saccharides and oligonucleotides under negative ionisation conditions. In all cases, the m/z scale must be calibrated by analysing a standard sample of a similar type to the sample being analysed (e.g. a protein calibrant for a protein sample), and then applying a mass correction.
5.3 Data processing
ESI and nanospray ionisation generate the same type of spectral data for samples, and so the data processing procedures are identical.
In ESI, samples (M) with molecular masses up to ca. 1200 Da give rise to singly charged molecular-related ions, usually protonated molecular ions of the formula (M+H)⁺ in positive ionisation mode, and deprotonated molecular ions of the formula (M-H)^- in negative ionisation mode.
An example of this type of sample analysis is shown in the m/z spectrum of the pentapeptide leucine enkephalin, YGGFL. The molecular formula for this compound is C₂₈H₃₇N₅O₇ and the calculated monoisotopic molecular weight is 555.2692 Da.
The m/z spectrum shows dominant ions at m/z 556.1, which are consistent with the expected protonated molecular ions, (M+H⁺). Protonated molecular ions are expected because the sample was analysed under positive ionisation conditions. These m/z ions are singly charged, and so the m/z value is consistent with the molecular mass, as the value of z (number of charges) equals 1. Hence the measured molecular weight is deduced to be 555.1 Da, in good agreement with the theoretical value.

Positive ESI-MS m/z spectrum of leucine enkaphalin, YGGFL.

The m/z spectrum also shows other ions of lower intensity (ca. 25 % of the m/z 556.1 ions) at m/z 557.2. These represent the molecule in which one ¹²C atom has been replaced by a ¹³C atom, because carbon has a naturally occurring isotope one atomic mass unit (Da) higher. The intensity of these isotopic ions relates to the relative abundance of the naturally occurring isotope multiplied by the total number of carbon atoms in the molecule. Additionally the fact that the ¹³C ions are one Da higher on the m/z scale than the ¹²C ions is an indication that z = 1, and hence the sample ions are singly charged. If the sample ions had been doubly charged, then the m/z values would only differ by 0.5 Da as z, the number of charges, would then be equal to 2.
The m/z spectrum also contains ions at m/z 578.1, some 23 Da higher than the expected molecular mass. These can be identified as the sodium adduct ions, (M+Na)⁺, and are quite common in electrospray ionisation. Instead of the sample molecules being ionised by the addition of a proton H⁺, some molecules have been ionised by the addition of a sodium cation Na⁺. Other common adduct ions include K⁺ (+39) and NH₄⁺ (+18) in positive ionisation mode and Cl^- (+35) in negative ionisation mode.
Electrospray ionisation is known as a "soft" ionisation method as the sample is ionised by the addition or removal of a proton, with very little extra energy remaining to cause fragmentation of the sample ions.
Samples (M) with molecular weights greater than ca. 1200 Da give rise to multiply charged molecular-related ions such as (M+nH)ⁿ⁺ in positive ionisation mode and (M-nH)^n- in negative ionisation mode. Proteins have many suitable sites for protonation as all of the backbone amide nitrogen atoms could be protonated theoretically, as well as certain amino acid side chains such as lysine and arginine which contain primary amine functionalities.
An example of multiple charging, which is practically unique to electrospray ionisation, is presented in the positive ionisation m/z spectrum of the protein hen egg white lysozyme.

Positive ESI-MS m/z spectrum of the protien hen egg white lysozyme.

The sample was analysed in a solution of 1:1 (v/v) acetonitrile : 0.1% aqueous formic acid and the m/z spectrum shows a Gaussian-type distribution of multiply charged ions ranging from m/z 1101.5 to 2044.6. Each peak represents the intact protein molecule carrying a different number of charges (protons). The peak width is greater than that of the singly charged ions seen in the leucine enkephalin spectrum, as the isotopes associated with these multiply charged ions are not clearly resolved as they were in the case of the singly charged ions. The individual peaks in the multiply charged series become closer together at lower m/z values and, because the molecular weight is the same for all of the peaks, those with more charges appear at lower m/z values than do those with fewer charges (M. Mann, C. K. Meng, J. B. Fenn, Anal. Chem., 1989, 61, 1702).
The m/z values can be expressed as follows:
m/z = (MW + nH⁺)/n
where m/z = the mass-to-charge ratio marked on the abscissa of the spectrum;
MW = the molecular mass of the sample
n = the integer number of charges on the ions
H = the mass of a proton = 1.008 Da.
If the number of charges on an ion is known, then it is simply a matter of reading the m/z value from the spectrum and solving the above equation to determine the molecular weight of the sample. Usually the number of charges is not known, but can be calculated if the assumption is made that any two adjacent members in the series of multiply charged ions differ by one charge.
For example, if the ions appearing at m/z 1431.6 in the lysozyme spectrum have "n" charges, then the ions at m/z 1301.4 will have "n+1" charges, and the above equation can be written again for these two ions:
1431.6 = (MW + nH⁺)/n and 1301.4 = [MW + (n+1)H⁺] /(n+1)

These simultaneous equations can be rearranged to exclude the MW term:
n(1431.6) - nH⁺ = (n+1)1301.4 - (n+1)H⁺
and so:
n(1431.6) = n(1301.4) +1301.4 - H⁺
therefore:
n(1431.6 - 1301.4) = 1301.4 - H⁺
and so:
n = (1301.4 - H⁺) / (1431.6 - 1301.4)

hence the number of charges on the ions at m/z 1431.6 = 1300.4/130.2 = 10.
Putting the value of n back into the equation:
1431.6 = (MW + nH⁺) n
gives 1431.6 x 10 = MW + (10 x 1.008)
and so MW = 14,316 - 10.08
therefore MW = 14,305.9 Da

The observed molecular mass is in good agreement with the theoretical molecular mass of hen egg lysozyme (based on average atomic masses) of 14305.14 Da. The individual isotopes cannot be resolved when the ions have a large number of charges, and so for proteins the average mass is measured.
This may seem long-winded but fortunately the molecular mass of the sample can be calculated automatically, or at least semi-automatically, by the processing software associated with the mass spectrometer. This is of great help for multi-component mixture analysis where the m/z spectrum may well contain several overlapping series of multiply charged ions, with each component exhibiting completely different charge states.
Using electrospray or nanospray ionisation, a mass accuracy of within 0.01% of the molecular mass should be achievable, which in this case represents +/- 1.4 Da.
In order to clarify electrospray/nanospray data, molecular mass profiles can be generated from the m/z spectra of high molecular mass, multiply charged samples. To achieve this, all the components are transposed onto a true molecular mass (or zero charge state) profile from which molecular masses can be read directly without any amendments or calculations.
The m/z spectrum of lysozyme has been converted to a molecular mass profile using Maximum Entropy processing and the data are shown. The mass profile is dominated by a component of molecular mass 14,305.7 Da, with a series of minor peaks at higher mass, which is usually indicative of salt adducting e.g. Na (M+23), K (M+39), H₂SO₄ or H₃PO₄ (M+98). The molecular masses can be read easily and unambiguously, and a good idea of the purity of the protein is obtained on inspection of the molecular mass profile.

Molecular mass profile of lysozyme obtained by maximum entropy processing of the m/z spectrum

Proteins in their native state, or at least containing a significant amount of folding, tend to produce multiply charged ions covering a smaller range of charge states (say two or three). These charge states tend to have fewer charges than an unfolded protein would have, due to the inaccessibility of many of the protonation sites. In such cases, increasing the sampling cone voltage may provide sufficient energy for the protein to begin to unfold and create a wider charge state distribution centering on more highly charged ions in the lower m/z region of the spectrum.
The differences in m/z spectra due to the folded state of the protein are illustrated with the m/z spectra of the protein apo-pseudoazurin acquired under different solvent conditions.
Analysis of the protein in 1:1 acetonitrile : 0.1% aqueous formic acid at pH2 gave a Gaussian-type distribution with multiply charged states ranging from n = 9 at m/z 1487.8 to n = 19 at m/z 705.3, centering on n = 15 (lower trace). The molecular mass for this protein was 13,381 Da. Analysis of the protein in water gave fewer charge states, from n = 7 at m/z 1921.7 to n = 11 at m/z 1223.7, centering at n = 9 (upper trace). Not only has the charge state distribution changed, the molecular weight is now 13,444 Da which represents an increase of 63 Da and indicates that copper is remaining bound to the protein. Many types of protein complexes can be observed in this way, including protein-ligand, protein-peptide, protein-metal and protein-RNA macromolecules.

Positive ESI-MS m/z spectra of the protein apo-pseudoazurin analysed in water at pH7 (upper trace) and in 1:1 acetonitrile:0.1% aq. formic acid at pH2 (lower trace).

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