1. INF380 - Proteomics-51 INF380 – Proteomics Chapter 5 – Fundamentals of Mass Spectrometry Mass spectrometry (MS) is used for measuring the mass-to-charge ratio of the components in a sample. The equipment used is called mass spectrometers. Very schematically, the mass spectrometers consist of three main parts: –the ionization source –the mass analyzer –the detector The components have to be ionized before their masses can be measured, and the ionization is performed in the ionization source. When dealing with peptides and proteins the ionization is commonly achieved by the addition of protons, hence the mass is also increased by the nominal mass of 1 Da times the number of charges The mass analyzer separates the components according to the mass-to- charge ratio (m/z) of the ions. Then the components hit the detector, and a mass spectrum is constructed by a connected computer The charge of a component must therefore be known before the mass can be determined. m/z is mostly considered dimensionless, but units as thomson (Th), u, or even Da are also used.

2. INF380 - Proteomics-52 Examples We have a peptide with mass 2000.0 Da, some of the peptide molecules get charge +1, others charges +2 and +3. The peptide ions will then be detected at –ions with charge +1: m/z = (2000 + 1)/1 = 2001 –ions with charge +2: m/z = (2000 + 2)/2 = 1001 –ions with charge +3: m/z = (2000 + 3)/3 = 666.7 The figure illustrates the main principle of mass spectrometry. A computer connected to the instrument constructs the mass spectrum. Suppose we have a sample of three peptides, with nominal masses A:680, B:481, C:400. Some of the A peptides get a charge of one and some of two, all B peptides get charge one, and all C peptides charge two. The mass/charge values for the ions become A:(680+1)/1=681 and (680+2)/2=341, B:482, C:201

3. INF380 - Proteomics-53 Ionization sources There are two main classes of mass spectrometers for proteomics, those that perform single mass spectrometry and those that perform tandem mass spectrometry (MS/MS). The former measures the m/z ratio of intact peptides. It is an advantage that the peptides mainly have a single (positive) charge. In tandem mass spectrometry the peptides are intentionally fragmented (into two fragments) to measure the masses of fragments. Often one wants to detect the mass of each fragment, therefore each have to be ionized. Thus, in tandem MS it is an advantage that the peptides carry several charges The two classes of instruments therefore often use different ionization sources, but exceptions exist. Some desired properties for the ionization process in proteomics are listed below. –All the components in the sample should be ionized in a detectable amount. –The ionized amount should be proportional with the sample component amounts. –There should be no fragmentation of the components unless we want to analyze the fragments. –There should be no unwanted adduct ions, –There should be no ions from other molecules (contaminants). There does not exist any ionization source that completely satisfies all these desires.

4. INF380 - Proteomics-54 Ionization sources MALDI (Matrix Assisted Laser Desorption Ionization) is the dominating ionization source for (single) MS –The matrix is small organic molecules that absorb light at specific wavelengths –The matrix is dissolved in an organic solvent under acidic conditions, and mixed with the sample. – A small drop is spotted on a sample plate, and the organic solvent evaporates. –During the evaporation, the matrix form small crystals, and the sample components are incorporated into the crystals. –A laser is then firing light (energy) onto the plate in very short pulses (a few nanoseconds), and the matrix absorbs the energy – Matrix molecules and sample molecules eject from the plate. The peptides are able to catch protons more or less well, and they become ionized. Most of the ionized peptides carry only one proton. –Under the influence of an electric field, the ions are transported to the mass analyzer. ESI (Electrospray Ionization) is primarily used for MS/MS. –The peptides are brought into the ionization source by a liquid flow, often from an HPLC. –The liquid is sprayed into a strong electromagnetic field, resulting in a mist of small droplets with a charged surface. –As the solvent in the droplets evaporate, the droplets get smaller and smaller, increasing the electric field on the surfaces. –When the electric field becomes strong enough, charged peptides desorpt from the surfaces. –Under these conditions, most of the ionized peptides will carry two protons, but higher charges are also often seen. –Under the influence of the electric field, the generated ions are transported to the mass analyzer.

5. INF380 - Proteomics-55 Mass analyzers There are several principles for mass analyzers used in proteomics, here we will briefly describe the simplest one to understand. The different analyzers are more described in following chapters. In the MALDI time-of-flight (TOF) mass analyzer ions are sent to the analyzer in short pulses due to the short laser pulses described above. The ions are accelerated by an electric field, and then they enter a field-free drift tube. The velocity that the ions have achieved during the acceleration is dependent on the mass and the charge of the ion, and this velocity is kept during the travel through the drift tube. Naturally, the time needed to pass the drift tube is dependent on the velocity. When the ions hit the detector at the end of the drift tube, the flying time is registered, and the m/z value can be calculated.

6. INF380 - Proteomics-56 Isotopic composition of peptides Many elements naturally exist in several isotopes. The isotopes are incorporated into molecules in a ratio that corresponds to their abundance in nature Six elements constitutes the overwhelming part of the elements of proteins Element Abundance % Mass –Hydrogen 1H 99.99 1.00783 – 2H 0.01 2.01410 –Carbon 12C 98.91 12.0000 – 13C 1.09 13.0034 –Nitrogen 14N 99.6 14.0031 – 15N 0.4 15.0001 –Oxygen 16O 99.76 15.9949 – 17O 0.04 16.9991 – 18O 0.20 17.9992 –Phosphorus 31P 100 30.9738 –Sulphur 32S 95.02 31.9721 – 33S 0.76 32.9715 – 34S 4.22 33.9676 Focus on isotopes of carbon, suppose that all the other elements are of the lightest isotope. Assume a small peptide with mass approximately M= 600 Da Such a peptide will contain around 30 carbon atoms. Approximately one third of the peptide molecules will therefore contain one 13C atom, the remaining being 12C. A very small amount of the peptides will contain two 13C atoms, thus approximately two thirds of the peptide molecules will only contain 12C As there is 1.0034 Da in mass difference between the peptide molecules only containing 12C and the peptide molecules containing one 13C atom and the remaining being 12C, the mass spectrum will show one high peak for the former peptide at mass M. This peak is called the monoisotopic peak. At mass M+1 there will be a peak of approximately one third of the intensity of the monoisotopic peak. A small peak may be seen at mass M+2, corresponding to the peptide with two 13C atoms. A peptide with mass approximately 3,000 Da, will have a low percentage of the peptide molecules that only contain 12C atoms.

7. INF380 - Proteomics-57 Isotopic composition of peptides

8. INF380 - Proteomics-58 Isotopic composition of peptides

9. INF380 - Proteomics-59 Isotopic composition of peptides A collection of isotopic peaks from the same peptide is called an isotopic envelope. Knowledge on the isotopic peak pattern helps to interpret mass spectra. We will mention two cases. Estimating the charge. –A singly charged peptide will have mass difference (m/z)=1 between the peaks in the isotopic envelope –Doubly charged peptides will have delta(m/z)=0.5 between the peaks, –and a triply charged peptide will have delta(m/z)=0.33 between the peaks of the isotopic envelope. At the same time, the m/z position of the peak in the mass spectrum is moved according to the formula (M + nH + )/nH +, where M is the mass of the peptide and n is the charge. This can be used to estimate the charge of the peptides.

10. INF380 - Proteomics-510 The raw data A detected compound is presented as a peak stretching over a (short) range of m/z values, rather than a single line at one exact m/z value (i.e., a peak without spreading). Thus for each peak there are numerous intensity measurements at defined small increments of the m/z value (defined by the time resolution of the detector), and these should be combined into one intensity value as a peak without spreading. Hence we can coarsely say that a mass spectrum can be presented in one of two forms, as raw data or as a peak list. The peak list is a processed form of the raw data. In its very simplest form, it contains a list of m/z values of the detected peptides. Most peak list formats, however, also includes the intensity value, and possibly other data, for each of the detected peptides. The intensity (the number of detected ions) is proportional to the area under the curve, and the physical value at the centroid of the peak isusually used to calculate the ion's m/z value. A typically raw data spectrum can for example contain about 3,000 pairs (m/z, intensity) inside a 100 unit interval, and the peak list containing four (corresponding to four peptides), but this depends on the instrument used.

11. INF380 - Proteomics-511 Resolution Resolution and resolving power in mass spectra are terms for describing the possibility to discriminate between different components with small differences in the masses. We will here present two definitions for resolution that are used to some extent.

12. INF380 - Proteomics-512 Resolution - p percent valley definition Let two peaks of equal height in a mass spectrum at masses m and m -  m be separated by a valley which at its lowest point is just p percent of the height of either peak. For similar peaks at a mass exceeding m, let the height of the valley at its lowest point be more (by any amount) than p percent of either peak height. Then the resolution (p percent valley definition) is m/  m. It is usually a function of m. The ratio m/  m should be given for a number of values of m. The common used value for p is 10.

13. INF380 - Proteomics-513 Resolution - peak width definition For a single peak made up of singly charged ion at mass m in a mass spectrum, the resolution may be expressed as m/  m, where  m is the width of the peak at a height which is a specified fraction of the maximum peak height. It is recommended that one of three values 50%, 5% or 0.5% should always be used. A common standard is the definition of resolution based upon  m being Full Width of the peak at Half its Maximum height, sometimes abbreviated `FWHM' (50%).

14. INF380 - Proteomics-514 Resolution Note that the percent valley definition depends upon two adjacent, mass spectral peaks of equal size and shape, which is rarely the case experimentally. So the resolution is commonly calculated from a single peak. However, for the most common peak shapes occurring in mass spectrometry, it is shown that for an isolated symmetrical peak recorded with a system which is linear in the range between x% and 2x% levels of the peak, the x% peak width definition is technically equivalent to the 2x% valley definition.