Mass Spectrometry: Peptide Sequencing
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1 Mass Spectrometry: Peptide Sequencing J Throck Watson, Michigan State University, East Lansing, Michigan, USA Electrospray and MALDI (matrix-assisted laser desorption ionization) are two versions of desorption/ionization that allow modern mass spectrometry to be applied to the analysis of peptides and proteins. In addition to a direct indication of the molecular weight, the sequence of amino acids in a peptide can be deduced from the fragmentation pattern (the mass spectrum itself). Introduction Mass spectrometric techniques for sequencing of peptides offer the potential for rapid and often automated analysis. In contrast to the chemical/chromatographic method of Edman degradation, the mass spectrometric techniques are applicable to N-terminally blocked peptides and they can provide direct evidence for the locus of posttranslational modifications, such as phosphorylation. The analysis consists of first converting the analyte into a charged species (ionization). The resulting ions are then desorbed into the gas phase, and some of them are degraded into fragments. Analysis of these ions according to their massto-charge ratio (m/z) provides considerable information. The m/z value for the residual intact, ionized molecules provides the molecular weight directly; mass analysis of the fragment ions allows the sequence of amino acid residues in the original peptide to be deduced. Ionization of Molecules of the Analyte Modern instrumentation developed during the 1990s (Hofstadler et al., 1996; Cotter, 1992) permits the efficient conversion of picomole ( mol), and sometimes femtomole ( mol), quantities of peptide to protonated molecules. Matrix-assisted laser desorption ionization (MALDI) is a batch sampling technique (i.e. not combined with a separation technique) that produces singly protonated molecules of the peptide. Electrospray (ES) most often produces multiply protonated molecules; ES can readily be combined with liquid chromatography, which allows for separation of components of a mixture of peptides prior to ionization. MALDI and ES have replaced fast-atom bombardment, which was the revolutionary ionization technique developed during the 1980s for analyses of nonvolatile, thermally labile substances, including peptides, at the nanomole ( mol) level. As both MALDI and ES are soft ionization techniques (i.e. they produce protonated molecules that undergo very little or no subsequent fragmentation because of the small. Introduction Secondary article Article Contents. Ionization of Molecules of the Analyte. Structural Information from Fragmentation of the Ionized Peptide. Promoting Fragmentation of the Ionized Peptide. Instrumental Techniques. Examples of Peptide Sequencing by Mass Spectrometry. Chemical Modification of the Peptide. Conclusion amount of energy imparted during the ionization process) they can be used to analyse mixtures of peptides because the mass spectrum of one peptide is unlikely to overlap with the spectrum of another. However, both MALDI and ES suffer from the problem signal suppression during the analysis of mixtures. For example, in principle, an equimolar mixture of four peptides would be expected to generate four protonated peptides of equal abundance to be represented in the mass spectrum by four peaks of equal intensity; in practice, if four peaks are observed during analysis of an equimolar quaternary mixture, they are unlikely to be of equal intensity. This phenomenon of signal suppression becomes more serious with increasing complexity of the mixture. Thankfully, although the mechanism of signal suppression is not fully understood, the problem in MALDI is frequently complementary to that observed in ES; further to analytical advantage, ES permits the use of liquid chromatography mass spectrometry (LC-MS) to reduce the complexity of the mixture entering the ion source at any given time, thereby minimizing problems with signal suppression. Structural Information from Fragmentation of the Ionized Peptide Ideally, cleavage of the ionized peptide at each peptide bond would provide a mass spectrum that could be interpreted, using a knowledge of the masses of the amino acid residues, to deduce the sequence. However, as illustrated conceptually in Figure 1, cleavage on either side of the a-carbon is also possible to give fragment ions that, while diagnostically useful, also complicate the spectrum (Roepstorff and Fohlman, 1984). Also note in Figure 1 that cleavage at any designated bond can generate either an N- terminal ion (a, b, c) or a C-terminal ion (x, y, z), the predominance of which for a protonated peptide (MH 1 ) depends on the locus of the more basic residues. In reality, the fragmentation process is more complicated than 1
2 H 2 N R 1 x 3 y 3 z 3 x 2 y 2 z 2 x 1 y 1 z 1 O R 2 O R 3 O R 4 C C N C C N C C N C COOH H H H H H H H a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3 Figure 1 Conceptual respresentation of key bonds in a given peptide that break to form the indicated N-terminal (a, b, c) or C-terminal (x, y, z) fragments. suggested in Figure 1; for example, creation of a y-ion involves hydrogen transfer from the N-terminal side of the peptide bond and retention of the ionizing proton. In addition, there can be fragmentation of the side-chain on certain residues; for example, fragmentation involving cleavage at the b-carbon of leucine and isoleucine generates w-ions, which distinguishes these two isomeric residues (Johnson et al., 1987). Recognizing the ion types as represented by the appearance of peaks in the mass spectrum is not critical, as most strategies for interpretation, especially those using an algorithm, involve an iterative computational approach. However, the beginning of a C-terminal series of fragments can be distinguished from the start of an N- terminal series. The largest b-ion will be represented by a peak at high m/z value that differs from that representing MH 1 by a number of mass units equal to the sum of the mass of an amino acid residue plus the mass of water, owing to expulsion of the C-terminal residue that contains the hydroxyl group. On the other hand, the largest y-ion is represented at a high m/z value by a peak differing from that for MH 1 by a number of mass units equal to only the mass of an amino acid residue. In principle, the sequence of a peptide could be deduced from a mass spectrum in which a complete series of any given ion type was represented. In practice, however, a complete series of any one type is rarely observed, but in fortunate situations overlapping patterns of two or more incomplete series may give complete sequence information. Ideally, one would prefer to observe complementary information from series of N-terminal and C-terminal fragment ions to bolster confidence in the analysis. Promoting Fragmentation of the Ionized Peptide The sequence of a peptide can be deduced from an interpretation of its fragmentation pattern (Kolli and Orlando, 1995). If an ionized peptide does not fragment sufficiently, it may not be possible to determine its sequence by mass spectrometry. Most peptides do not fragment immediately upon ionization; however, depending upon the energetics of ionization and the kinetics of decomposition, a portion of these ionized peptides may fragment as a function of time after ionization (within s). The fragmentation pattern resulting from this metastable decay of ionized peptides can be measured with a time-offlight mass spectrometer (equipped with a reflectron) after ionization by MALDI; the technique is called MALDI post-source decay, where ions decompose in the flight tube en route to the reflectron/detector. Frequently, a better yield of fragmentation can be achieved through collisionally activated dissociation. Instrumental Techniques Collisionally activated dissociation (CAD) differs from post-source decay (PSD) in that the protonated molecules are caused to decompose from additional energy that they gain upon collision, rather than decomposing owing to the energy they obtain at ionization. CAD provides a means of increasing the internal energy of an ion to promote fragmentation (or dissociation). Through collisions, some of the kinetic energy (KE) of an ion is converted into internal energy; the higher the KE of the ion or the closer in mass are the colliding particles, the greater the conversion of KE to internal energy. Thus, a 2-kDa ion with KE 5 1 kev will fragment more efficiently after colliding with argon (40 Da) than with helium (4 Da). Ion molecule collisions are used in CAD because of the ease of controlling the pressure of the collision gas, and the neutral molecules provide no complications of electrostatic interactions with the ion of interest. While CAD can be used with ions formed by any process, in the analysis of peptides it is most often employed with the electrospray. The classical arrangement for CAD involves mass spectrometry/mass spectrometry (MS/MS) in which one m/z analyser (MS1) is placed ahead of a collision cell (in which the pressure of a collision gas might be torr, as opposed to torr in the analyser), and the second m/z analyser (MS2) follows it. In this way, MS1 is used to select the ions of a given m/z value that are allowed to enter the collision cell, and MS2 is used to analyse the products of CAD. A less expensive, but also less selective, version of CAD can be achieved by adjusting electric fields near the ion source. The cone voltage in an electrospray interface controls the electric field between the ionization region and the cone, which contains an orifice through which the ions enter the vacuum system of the m/z analyser. In this less selective version of CAD, called in-source CAD, all the ions (not just selected ones as in MS/MS) are accelerated in the electric field controlled by the cone voltage to collide with residual molecules. With in-source CAD, the degree of fragmentation is directly related to the magnitude of the cone voltage. In-source CAD is usually a lower-energy 2
3 process than CAD accomplished in an MS/MS collision cell. Examples of Peptide Sequencing by Mass Spectrometry The mass spectrum of LKRApTLG-amide as analysed by ES-CAD-MS/MS is shown in Figure 2. Ions of the peptide were produced by ES to form both single- and doubleprotonated molecules. The ions in the form of singleprotonated molecules (MH 1 ) were selected by MS1 for entry into the collision cell. Following CAD with helium in the collision cell, the product ions were analysed by MS2 to generate the mass spectrum shown in Figure 2. A cursory examination of Figure 2 indicates that a variety of ion types are represented in the mass spectrum. For example, the peak labelled b 3 represents the first three residues from the N-terminus; d 6 represents the first six residues from the N-terminus after loss of the side-chain of Leu at the b-carbon; and y 5 represents the first five residues from the C-terminus. The difference in m/z values of peaks representing any two adjacent members of a series of ions equals the mass of the residue corresponding to that site. For example, b 6 is represented by the peak at m/z and b 5 is represented by the peak at m/z 650.4; the difference between the two, , corresponds to the residue mass of Leu (113 Da). Depending upon the question being asked about a putative peptide, analysis by mass spectrometry can provide considerable insight regarding its structure. For example, if the analyte were thought to be a peptide having the sequence LKRATLG-amide (or some other permutation of these residues), its calculated molecular weight would be Da. Analysis of such a peptide by ES-MS would be expected to produce an abundant protonated molecule (MH 1 ) represented by a peak at m/z (data not shown). On the other hand, if the ES-MS spectrum showed a peak at m/z (80 mass units higher than expected), such information (data not shown) would suggest that the putative peptide (LKRATLG-amide) contained a phosphate group (80 Da). Such an ES-MS spectrum would not indicate the location of the phosphate within the peptide. An ES-CAD-MS/MS spectrum of the ion current at m/z could provide this information; in fact, the ES-CAD-MS/MS (of the ion current at m/z ) shown in Figure 2 confirms the sequence of the analyte as LKRApTLG-amide and indicates the location of the phosphate group (on threonine, T). The peak for b 5 in Figure 2 appears at m/z and that for b 4 appears at m/z 469.4; the difference between the two is 181 mass units. The mass of a threonine residue is 101 Da and the mass of a phosphate group is 80 Da; thus, the threonine residue is phosphorylated as indicated by the lower case p (pt) in the single-letter code sequence for the peptide. In this didactic example, threonine is the only residue capable of being phosphorylated, but in more complex cases, in which there are multiple possible sites for phosphorylation, the same strategy for data interpretation is used. In the mass spectrum in Figure 2, none of the ion series is complete, but upon iterative consideration of many peaks representing the indicated ion types, the sequence can be deduced. This spectrum may not be easy for the novice to b 3 b 3 -NH 3 b 4 b 4 -NH 3 y 4 -H 3 PO 4 y 5 -H 3 PO 4 b 6 -H 3 PO 4 Relative intensity b 5 -NH 3 y 5 Mass Spectrometry: Peptide Sequencing b 5 d 6 b 6 MH-H 2 O + MH + MH + -H 3 PO m/z Figure 2 ES-CAD-MS/MS mass spectrum of a peptide, LKRApTLG-amide. 3
4 interpret, but it is sufficient for data processing by proven algorithms (Yates et al., 1995), especially if the amino acid composition of the peptide is known. Even though there is some degeneracy in the masses of some residues (e.g. both lysine and glutamine, as well as the combination of the two residues GlyAla, have a mass of 128 Da), the algorithm can successfully automate the interpretation process (Yates et al., 1995). Chemical Modification of the Peptide Frequently, conversion of the peptide to a charged derivative before analysis by MS greatly simplifies the mass spectrum, and facilitates its interpretation (Shen et al., 1999). Figure 3 shows the ES-CAD-MS/MS of the charged derivative of LKRApTLG-amide (in this case, the N- terminus of the peptide has been converted to the acetyltris(trimethoxyphenyl)phosphonium (Ac-TMPP) derivative, in which the phosphorus contains a fixed charge). The fixed charge at the N-terminus of the peptide stimulates cleavage at nearly every peptide bond (via a remote-site mechanism) to generate a complete series of *a-type ions, as indicated by the labelled peaks in Figure 3. The Ac-TMPP group adds 472 Da to the mass of the peptide; the molecular cation is represented by the peak labelled C 1 (the fixed charge on the quaternary phosphorus in Ac-TMPP converts the peptide molecule to a cation) in Figure 3. The difference in mass units between the fragment ion peaks corresponds to the mass of the amino acid residues lost from the C-terminus during the remotesite fragmentation process. The sequence of amino acids starting from the C-terminus can be read off the mass spectrum starting with the first high-mass fragment. For the most part, fragment ions from a protonated underivatized peptide (as in Figure 2) are generated by cleavage at a protonated amide; the site of the protonated amide is influenced by the relative proton affinity of the various amino acid residues. The variety of ion types found in the spectrum of an underivatized peptide (Figure 2) is explained by the mobile-proton model in which the proton is captured by the most basic residues, and the locations of those basic residues influence whether N- terminal or C-terminal ions predominate. The remote-site mechanism is apparently more uniform in nature, as suggested by the appearance of a more complete series of N-terminal ions represented in Figure 3. Conclusion ES and MALDI are two versions of desorption/ionization that allow modern mass spectrometry to be applied to the analysis of peptides and proteins. While both ES and 10 Relative intensity Ac-TmPP *a 1 6 *b 1 *c 1 *b *a 3 -NH *b m/z *b *b 5 -H 3 PO *a 5 *b *d *a 6 -NH C + -H 3 PO 4 C + -NH *b *c 6 C Figure 3 ES-CAD-MS/MS mass spectrum of Ac-TMPP derivative of LKRApTLG-amide. The charged derivative (Ac-TMPP) of the peptide promotes remotesite fragmentation, which gives a more complete series of fragment ions (compared to that in Figure 2) from which the sequence and locus of the phosphate group can be deduced. 4
5 MALDI are soft ionization processes, each can be combined with instrumental techniques, such as CAD and PSD, respectively, to promote fragmentation of the protonated molecules. Analysis by mass spectrometry can provide considerable insight regarding the structure of peptides and proteins. In addition to a direct indication of the molecular weight, the sequence of amino acids in a peptide can be deduced from the fragmentation pattern (the mass spectrum itself). References Cotter R (1992) MALDI-TOF-MS. Analytical Chemistry 64: 1027A 1039A. Hofstadler SA, Bakhtiar R and Smith RD (1996) Instrumentation and spectral interpretation for electrospray MS. Journal of Chemical Education 73: A82 A88. Johnson RS, Martin SA, Biemann K, Stults JT and Watson JT (1987) Novel fragmentation process of peptides by CAD-MS/MS: differentiation of leucine and isoleucine. Analytical Chemistry 59: Kolli VSK and Orlando R (1995) Complete sequence confirmation of large peptides by high energy CAD. Journal of the American Society of Mass Spectrometry 6: Roepstorff P and Fohlman J (1984) Nomenclature for MS of peptides. Biomedical Mass Spectrometry 11: 601. Shen T-L, Huang Z-H, Laivenieks M et al. (1999) Evaluation of charge derivatization of a proteolytic digest for improved mass spectrometric analysis. Journal of Mass Spectrometry 34: Yates JR, Eng JK and McCormack AL (1995) Correlating MS/MS of peptides to sequences in nucleotide databases. Analytical Chemistry 67: Further Reading Watson JT (1997) Introduction to Mass Spectrometry, 3rd edn. Philadelphia: Lippincott-Raven. 5
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