A mass spectrometer consists of an ion source, a mass analyzer that measures the mass-to-charge ratio (m/z) of the generated ions, and a detector that detects the number of ions at each m/z value. Two suitable techniques for the ionization of peptides and proteins are ESI and MALDI (see above). The analytes are ionized out of a solution (ESI) or out of a crystalline matrix via laser pulses (MALDI). Whereas these two techniques are set, a wider range of mass analyzers is available. Important parameters are sensitivity, resolution, mass accuracy, and the ability to generate information-rich mass spectra. The basic types of mass analyzers currently used in proteomic studies are time-of-flight (ToF), ion trap, quadrupole, Fourier transform ion cyclotron resonance (FT ICR), and the Orbitrap mass analyzers.
MALDI is usually coupled to ToF analyzers whereas all other mass analyzers are commonly coupled to an ESI source. In addition, hybrid mass analyzers, such as hybrid quadrupole ToF
(Qq-ToF) mass analyzers, are available. Here, precursor ions for MS/MS experiments are selected in the first quadrupole, fragmented in a collision cell, and the fragment ion masses are analyzed in the ToF. These instruments are commonly coupled to an ESI source and have high sensitivity, resolution, mass accuracy, and - most important - generate information-rich fragment spectra.
Quantitative information can be obtained from MS or MS/MS signals (see above). The advantage of quantification from MS spectra is that several spectra are available and ion intensities are high. However, very low and very strong signals are problematic. Low signals are hard to distinguish from background noise and very strong signals can saturate the detector, what in turn limits precision of the measurement. The latter is more often observed in ToF and Qq-ToF instruments compared to ion traps as the latter can control the number of ions before detection. Using fragment ion intensities (MS/MS) for quantification, detector saturation and interference with background ions can be neglected. Low intensities are rather a problem as poor ion statistics may result in less robust quantification. However, limits to quantification of complex samples can often be attributed to interference of co-eluting components of similar masses.
Different specific mass spectrometric scanning modes are used to read out signal intensities during quantitative analysis. The commonly used and most powerful techniques will be described in the following paragraphs.
MALDI-ToF-MS Technical advances have enhanced the application of MALDI mass spectrometry for proteomics but also for quantitative studies. The investigation of tandem time-of-flight instruments (ToF-ToF) allows the fragmentation of precursors and thus unambiguously assigns the species to be quantified (Bienvenut et al., 2002). Decoupled MS and MS/MS analysis allows for data-dependent MS/MS analysis, and the manner of sample preparation reserves most of the sample for repeated analysis. Furthermore, the generation of predominant singly charged ions during MALDI simplifies data analysis (Pan et al., 2009b).
During MALDI-ToF-MS quantitative information is often obtained from the area under the peaks to be quantified. However, LC-offline allows the generation of extracted ion chromatograms (XICs, see below) over the whole chromatographic timescale for the peptide of interest. However, in a MALDI spectrum there is often a large discrepancy between ion intensities and analyte concentration on the MALDI target. Ionization of the peptides occurs via proton transfer from the acidic matrix and the ionization efficiency is therefore dependent on the proton affinity of the different peptides. The presence of a peptide with a very high proton affinity can consequently influence the intensity of other ions through ion suppression effects (Knochenmuss et al., 2000). For this reason, peptides below a mass of 3 kDa can only be quantified using standard peptides, which have the same chemical structure and thus
show the same behavior during the ionization process (see Figure 2.3 for an example of a spectrum of an endogenous and a standard peptide acquired by MALDI-ToF-MS). Peptides or proteins larger than 3 kDa have such a high proton affinity that ion suppression effects are very unlikely (Wilm, 2009).
Extracted ion chromatograms (XICs) Electrospray ionization is well suited for quantitative measurements if the flow rates are 100 nl/min or lower. In this case, spectral intensities correspond to the analyte concentrations very well. For higher flow rates the electrospray is unsteady and ion intensities become irregular and do not reflect molecular concentrations (Wilm, 2009). In addition, ion suppression has been observed for higher flow rates (Schmidt et al., 2003). The peptide’s signal in the MS analysis can be plotted over time while the peptide is eluting from the chromatography column, i.e. so-called extracted ion chromatograms (XICs) for defined peptides can be generated. The XIC signal is related to the relative amount for the same peptide at the same experimental conditions and can therefore be used for comparison of the same peptide in different samples. XICs are usually generated from samples analyzed by LC-ESI-MS whereas generation of XICs from LC-offline MALDI-MS analyses is also possible (see above). Using high mass accuracy mass spectrometers, greater than two-fold changes of a peptide can be measured. As one peptide in a complex mixture is not always selected for fragmentation in different MS runs (Kuster et al., 2005) it is critical to find and quantify the correct peptide in the different analyses to be compared. Development of required software and normalization of the runs to be compared by spiked-in calibrants can overcome this problem. The great advantage of XIC-based quantification is that no labeling strategy is used and almost every different MS analyses can be compared as long as they were performed under the same conditions. However, this requires a very high reproducibility during sample preparation, chromatography, and MS analysis. One alternative to circumvent this problem is the use of stable isotope labeled standard peptides in known amounts. As the labeled and the non-labeled peptide show the same behavior during chromatography and MS generated XICs from both peptides show the same chromatographic retention time. The area under the XICs can then be used for absolute or relative quantification (see Figure 2.7 for an example of XICs of a labeled and a non-labeled peptide).
Figure 2.7: Example of Extracted ion chromatograms (XICs) to read out the signal intensities for quantification. (A) Total ion count of the hydrolyzed hPrp19/CDC5L complex. (B) MS spectrum of ILLGGYQSR (CDC5L protein) and the stable isotope labeled standard peptide. According to the incorporated stable isotope labeled amino acid (arginine) a mass difference of 5 m/z between the doubly charged peptides is observed. (C) Extracted ion chromatogram of ILLGGYQSR. (D) Extracted ion chromatogram of the standard peptide ILLGGYQS(R). The endogenous and the standard peptide show the same retention time. The peak area of the signals can be used for quantification.
Multiple Reaction Monitoring (MRM) A quadrupole mass analyzer can be operated as a mass filter allowing only one specific m/z to pass the quadrupole. This feature is utilized to detect the specific transition from a given precursor to a user-defined fragment ion (selected reaction monitoring, SRM) in a triple quadrupole mass spectrometer. To this end, the precursor mass is selected in quadrupole Q1, whereupon (after fragmentation in q2) the user-defined fragment ion is detected in Q3 (SRM transition). This technique has been extended to the detection of multiple fragment ions per precursor and is then called multiple reaction monitoring (MRM; Anderson and Hunter, 2006; Kuhn et al., 2004; Stahl-Zeng et al., 2007, Figure 2.8). The MRM signal is quantitative over 4-5 orders of magnitude (Wolf-Yadlin et al., 2007) and can be used for relative and for absolute quantification as outlined in section 2.1.3. One run, in which all MRM transitions are monitored once, is called a duty cycle and its length is dependent on the dwell time (i.e. the time to accumulate ions in the quadrupole), the
number of precursors, and the number of MRM transitions per precursor. The duty cycle is repeated consistently during the MRM analysis. It should be repeated several times while the peptide is eluting from the chromatography column to achieve a certain number of data points, which are required to record a sufficient MRM signal. The length of the duty cycle is therefore not unlimited and needs to be adjusted for every analysis. MRM is a very sensitive and, as two mass filters are connected in series, a very specific method. For this reason, it is not only a quantification technique but also very well suited for targeted proteomics, such as biomarker verification and validation.
Figure 2.8: MRM workflow. A peptide mixture containing endogenous and stable isotope labeled standard peptides is analyzed by MRM in a triple quadrupole mass analyzer. In quadrupole 1 (Q1) a defined precursor ion is selected and, upon fragmentation in q2, a specific fragment ion of the selected precursor is detected in Q3.
After a defined time (e.g. 20 ms) the next precursor is selected for MRM analysis. One duty cycle comprises the analysis of all MRM transitions scheduled for the peptide sample to be analyzed. The obtained signal for the MRM transitions is quantitative and can be used for relative and absolute quantification of the peptides.
Parallel fragmentation (MSE) A very different approach to analyze and quantify peptides is parallel fragmentation (also known as MSE; Silva et al., 2006). Here, all precursors, entering the mass spectrometer at a particular time, are fragmented without selecting an individual precursor ion. For this purpose, the mass spectrometer switches continuously between MS and MS/MS mode, thus delivering an almost complete data set of the sample. Fragment ions are assigned retrospectively to their precursors by their identical time profiles. However, this requires a well-resolved chromatographic system and the assignment of fragment ions to their precursor might fail due to fragment ions that are generated by several precursors simultaneously. Therefore, the MSE scanning method is performed on Qq-ToF mass spectrometers that might compensate probable
mis-assignments by their high mass resolution in MS/MS. Once the experimental data are obtained, the data set can be interrogated for a specific ion and a set of specific fragment ions (pseudo-MRM; Niggeweg et al., 2006). This interrogation is not as specific as a real MRM experiment but has been proven for direct, label-free quantification (Wilm, 2009).