Nominal mass determinations
Exact mass (150-1000 Da)
High mass accuracy of peptides
ESI of oligonucleotides
ESI of proteins
Multiply charged ions
ESI data on the QTof
Dr Alison E. Ashcroft, University of Leeds
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Electrospray ionization mass spectrometry (ESMS) is carried out on the Micromass Q-Tof mass spectrometer on samples with molecular weights from 100 to well over 100,000 (More Information). In addition, many other types of samples (e.g., carbohydrates, lipids, and polymers) may also be analyzed on the Q-Tof.
It is important to note also that the average mass accuracies given below are estimated and that the mass error on any given sample may be either higher or lower than these estimates. Monoisotopic masses should be used for molecules with mass less than 3 kDa while average masses should be used for larger molecules.
This type of analysis is extremely useful for rapid monitoring of chemical syntheses where an average mass error of +- 0.2 amu (400 ppm or +- 0.04% for 500 Da) is sufficient to determine if the synthesis is indeed proceeding as planned. This level of mass accuracy can be achieved routinely on the Q-Tof with external calibration and without having to constantly monitor instrument calibration.
To reach the extremely high level of mass accuracy required to confirm the elemental composition of the products of chemical syntheses requires the use of internal calibrants. To avoid signal suppression, the concentration of these internal calibrants have to be carefully matched to that of the sample. The average mass accuracies that may be achieved with this approach are +- 0.002 Da over the mass range 150-400 and 5 ppm (+- 0.0005%) over the mass range extending from 400-1000)
Frequent monitoring of the external calibration of the Q-Tof allows the average mass error with external calibration to be decreased to less than +-0.05 amu (50 ppm or +-0.005% for 1000 Da). The level of mass accuracy achieved with this approach would be useful for identifying proteins via peptide mass database searching where the higher mass accuracy will increase the specificity of the identification. In this instance the analysis typically would be carried out on an in gel tryptic digest of an SDS-PAGE separated, Coomassie Blue stained gel band that had been subjected to a reverse-phase "clean-up" on a ZipTip. The resulting peptides would be analyzed using nanospray which would require about 2 ul of a >500 fmol/ul solution.
ESMS spectra of oligonucleotides are recorded in negative ion mode. To increase sensitivity and mass accuracy the samples should be free of sodium and potassium counterions. There are many published methods to remove or minimize the effect of these counterions. These include organic precipitation, RP-HPLC, ZipTip clean-up (which also rellies on a reverse phase support), or carrying out the ESMS in the presence of triethylamine acetate, piperidine, or imidazole buffers. We use the latter two methods: ZipTip clean-up and/or one of the three buffers mentioned. The longest oligonucleotide we have analyzed was a 39-mer. ESMS spectra of longer oligos have been reported in the literature. We request that a minimum of a 100 pmol be submitted. Any unused sample can be returned if requested beforehand.
For large molecular weight biomolecules (e.g., proteins), it is important to recognize that the measured mass is the average mass and that the peak envelope extends over many individual masses. For example, a protein with a mass of 10 kDa. will have a peak envelope that is approximately 20 mass units wide (counting all isotope containing peaks with intensities greater than 1% of the most abundant peak, for a detailed discussion see Anal. Chem. 55,353-356 (1983)). As the molecular size of the molecule increases, the peak envelope gets wider such that the envelope for a 100 kDa protein should contain 57 molecular ion peaks. In instruments that cannot resolve these individual isotopic peaks, the width of this isotopic envelope will determine what mass difference is needed between two molecules in order to detect each molecule. As a general guide, one should be able to theoretically detect two components present in equal amounts if their molecular weight divided by their mass difference is less than approximately 1,000. Hence, if the samples are very clean (i.e., no counterions or adducts which would create 'tails' on the mass spectral peaks) it may be possible to detect a 50,000 Da species in the presence of an equivalent concentration of a 50,050 dalton species.
ESMS spectra are almost always recorded in positive ion mode. The mass spectrum from a protein contains a series of peaks produced from the protein by adding a successive number of protons. Because mass spectrometers record mass-to-charge ratio, electrospray analyses on proteins typically produce a spectrum in the mass range of 500-2000. This spectrum containing multiply charged peaks is processed to produce a peak on a molecular mass scale by either transforming manually identified multiply charged peaks (adjacent peaks can only differ by one charge) or by MaxEnt transformation which only requires an accurate peak width as input. The MaxEnt transformation produces a mock spectrum which can be compared to the raw data to determine the quality of the transformation. The average, expected mass error is about +- 0.01%. (More Information).
Although we sometimes see +2 charged species during MALDI-MS, short peptides tend primarily to give just the +1 species so this makes it easy to directly interpret MALDI-MS spectra. The problem is that all types of MS actually measure the mass/charge ratio (m/z) as opposed to the mass. Hence the following two scenarios give identical spectra with a single observed peak (in positive ion mode) at m/z of 2,001:
Actual (M) Peptide mass = 2,000, Charge = +1,
Observed (M+H) m/z = (2,000 + 1)/1 = 2,001
Actual (M) Peptide mass = 4,000, Charge = +2, Observed (M+H) m/z = (4,000 + 2)/2 = 2,001
Multiple charge states are potentially a severe problem for three of our electrospray ionization mass spectrometers, the LCQ and Q-Tof.. Instead of the single (M+H) species characteristic of linear MALDI-MS, electrospray usually gives a broad spectrum of multiply charged ions for each species present, which is why a mass spectrometer equipped with an electrospray source that has an upper m/z limit of 1,800 can easily determine the m/z for a 50,000 dalton protein. Because of multiple charging many m/z ratios obtained from the electrospray ionization mass spectrometers do not correspond with actual peptide masses.
There are two general methods for processing electrospray mass spectra into their singly charged format on the Q-Tof mass spectrometer, namely, the transform method and the Maximum Entropy method. The method selected depends on the quality of the raw data (e.g., signal to noise ratio and the number of components present in the sample). The transform method is the preferred method if the component peaks in the multiply charged raw spectrum can be identified. This method provides multiple measurements of the molecular weight because each multiply charged ion is an independent measurement of the molecular weight. From these multiple measurements an average mass can be calculated and a standard deviation. These multi-charged spectra can then be transformed into the equivalent singly charged spectrum. In this instance, the sample submitter would receive three types of data, the list of the multi-charged masses and the calculated molecular weight, a plot of the transformed, singly charged spectrum, and a plot of the multi-charged spectrum. The major limitation of this method is the appearance of artifact peaks in the transformed spectrum from background ions. These are usually readily apparent however because they do not have other related component peaks.
Maximum Entropy processing is the only method that can be used to process multiply charged spectra when adjacent component peaks cannot be identified because of low signal or because multiple components in the sample hinder the identification. The only required input for Maximum Entropy processing is the expected molecular mass range of the compound of interest and an estimate of the peak width for a compound of that mass range with the number of charges needed to produce the observed multi-charged spectrum. This latter parameter takes into account the width of the isotope envelope and instrument resolution. The output from a maximum entropy processed spectrum is a plot showing the singly charged molecular ion and a reconstructed multi-charged spectrum from MaxEnt which can be compared to the multi-charged raw spectrum which then provides a measure of the quality of the input parameters. When MaxEnt is used sample submitters would receive copies of all three outputs.
Spectra processed with the transform algorithm have in addition to the list of masses, the letters Tr in the second line in the upper left hand corner of the molecular mass spectrum. Maxent spectra contain in addition to the reconstructed multicharged spectrum the letters Mk in the second line of the molecular mass spectrum.
Dr Alison E. Ashcroft, University of Leeds
