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B ac k t o B a sic s Peptides and Proteins Analysis by MS Back to Basics Section G: Applications CHAPTER G5 PEPTIDES AND PROTEINS ANALYSIS BY MS TABLE OF CONTENTS Quick Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591 Fast Atom Bombardment (FAB) . . . . . . . . . . . . 591 Dynamic FAB . . . . . . . . . . . . . . . . . . . . . . . . . . 593 Mass Spectrometry Mass Spectrometry (MS-MS) . . . . . . . . . . . . . . 593 Other Ion Sources . . . . . . . . . . . . . . . . . . . . . . . 595 Laser Desorption Mass Spectrometry (LDMS) . 597 Electrospray (ES) . . . . . . . . . . . . . . . . . . . . . . . 597 Proteins of Large Molecular Mass . . . . . . . . . . . 601 Analysis of Peptide Mixtures . . . . . . . . . . . . . . . 603 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 Mic rom as s U K Limite d Page 585 B ac k t o B a sic s Peptides and Proteins Analysis by MS This pa ge is intentiona lly b lan k. Mic rom as s U K Limite d Page 586 B ac k t o B a sic s Quick Guide Peptides and Proteins Analysis by MS • Amino acids are the molecular building blocks of peptides and proteins. About 20 common amino acids are known. • Peptides and proteins are formed by linking successive amino acids into chains or rings. The order (sequence) and types of amino acids determine the chemical and physical properties of peptides and proteins. • An enzyme is a special protein which acts as a catalyst for biochemical reactions. • Fast Atom Bombardment (FAB) is an ionization technique which produces a protonated or deprotonated molecular ion and hence a molecular mass for the sample. • Liquid Secondary Ion Mass Spectrometry (LSIMS) is a similar but more recent technique than FAB, and produces indistinguishable data. • Dynamic/Continuous Flow FAB allows a continuous stream of liquid into the FAB source, and hence constitutes an LC-MS interface. • An enzyme digest is the term applied to a process whereby a peptide or protein is mixed with a selected enzyme under favourable conditions to allow reaction to occur. The enzyme splits the peptide or protein into smaller units which are easier to identify. • Post-translational modifications to proteins are biochemical in origin and alter the measured molecular mass relative to that calculated for an untranslated sequence. • Laser Desorption Mass Spectrometry (LDMS) coupled to a Time-of-Flight analyser produces an unresolved protonated or deprotonated molecular ion cluster with virtually no upper mass limit. • Electrospray (ES) produces a series of multiply charged ions which is transformed into an accurate molecular mass profile with virtually no upper mass range limit. • Peptides and proteins can be analysed by mass spectrometry. Molecular mass information can be found by FAB or LSIMS for samples up to 10 kDa in mass. Laser Desorption and electrospray can analyse much higher molecular mass samples. Mic rom as s U K Limite d Page 587 B ac k t o B a sic s Peptides and Proteins Analysis by MS 556 100% 90 Tyr-Gly-Gly-Phe-Leu MW = 555 80 556 = [M+H]+ 70 60 50 40 30 397 20 10 336 317 0 300 Figure 1 Mic rom as s U K Limite d 350 425 400 450 500 550 600 mass 650 For this FAB experiment, a sample of the pentapeptidic enkephalin, Tyr.Gly.Gly.Phe.Leu., dissolved in glycerol was bombarded by xenon atoms. The resulting mass spectrum shows abundant protonated molecular ions at m/z 556. Page 588 B ac k t o B a sic s Peptides and Proteins Analysis by MS • Tandem Mass Spectrometry (MS-MS) produces precise structural or sequence information by selective and specific induced fragmentation, routinely on samples up to 2500 Da. For samples of greater molecular mass than this, an enzyme digest will usually produce several peptides of molecular mass suitable for mass spectrometry. • Samples containing mixtures of peptides can be analysed directly by electrospray. Alternatively they can be separated and analysed by LC-MS coupling techniques such as Dynamic/Continuous Flow FAB, or Electrospray. Summary Mic rom as s U K Limite d The use of mass spectrometry for the analysis of peptides, proteins and enzymes has been summarized. This guide should be read in conjunction with others in the ‘Back to Basics’ series including ‘Biotechnology’ and those describing specific ionization techniques in detail. Page 589 B ac k t o B a sic s Peptides and Proteins Analysis by MS 5 [ 1 ; R = acetyl 1) Acetyl 2) 8-hydroxybutyryl 3) Propionyl 4) Crotonyl 5) Butyryl + Isobutyryl 6) Isovaleryl 7) Octanoyl ] 6 1 3 4 2 4:4 8:2 7 12 15:4 TIME (min) (a) NH 2 N N 303 - = = O NH OH CH3 - - - = O - - CH3 OH OH C - CH2 - CH2 - NH - C - CH - C - CH2 - O - P - O - P - O - O = O 508 CH2 N 136 CH2 O - 428 O OH - S HO - P - OH - = - CH2 136 N 330 R O ( 1; R = acetyl) 303 MH+ 810 428 508 330 100 500 800 (b) Figure 2 Mic rom as s U K Limite d (a) LC-FAB-MS analysis of short and medium chain acyl coenzyme A compounds (1, = 1-7; 0.5 nmol of each). These compounds carry acyl groups for enzyme reactions and a number of metabolic diseases can be traced to enzyme deficiencies which result in the accumulation of toxic coenzyme A thioesters. (b) The FAB mass spectrum of acetyl coenzyme A (component 1.1 from the trace shown in (a). The likely origin of major fragment ions is indicated. Page 590 B ac k t o B a sic s Peptides and Proteins Analysis by MS ANALYSIS OF PEPTIDES AND PROTEINS BY MASS SPECTROMETRY Introduction Until 1981, mass spectrometry was limited, generally, to the analysis of volatile, relatively low molecular mass samples and was difficult to apply to involatile peptides and proteins without first cutting them chemically into smaller volatile segments. During the past decade the situation has changed radically with the advent of new ionization techniques and the development of tandem mass spectrometry. Now, the mass spectrometer has a well-deserved place in any laboratory interested in the analysis of peptides and proteins. Fast Atom Bombardment (FAB) The first breakthrough came with the development of FAB which enabled polar compounds of large molecular mass to be ionized without application of heat for volatilization of the sample. In FAB (see ‘Back to Basics’ guide), the sample is dissolved in a suitable solvent (also called a matrix) of low volatility (e.g. glycerol, thioglycerol, m-nitrobenzyl alcohol) and is bombarded by a beam of fast xenon or argon atoms. Ionization produces protonated [M+H+] or deprotonated ([M-H]-) molecular ions, sometimes accompanied by a little fragmentation. The matrix reproducibly gives background ion peaks but these can interfere with sample ion peaks and usually dominate the low mass end of the spectrum (Figure 1). Different samples exhibit different levels of response to FAB and, with a mixture of components, it is feasible that not all will be detected; in some cases, the minor components of a sample appear more prominently in the mass spectrum than the major ones. Despite these limitations, FAB is in widespread use and is an excellent technique for determining the molecular masses of peptides up to 10,000 Daltons, with an accuracy of 0.5 Da. FAB has evolved and fast atoms are being replaced by fast ions, such as Caesium (Cs+). This variation is called Liquid Secondary Ion Mass Spectrometry (LSIMS) because the sample solution affords the secondary ion beam whilst the bombarding ions constitute the primary beam. Spectra produced by FAB and LSIMS are virtually identical, although higher sensitivity at high mass (10 kDa) is claimed for the latter. Mic rom as s U K Limite d Page 591 B ac k t o B a sic s Peptides and Proteins Analysis by MS ION SOURCE Figure 3 Mic rom as s U K Limite d MS(1) CC MS(2) A typical MS-MS configuration. Ions produced from a source (e.g. dynamic-FAB) are analysed by MS(1). Molecular ions (M+ or [M+H]+ or [M-H]- etc.) are selected in MS(1) and passed through a collision cell (CC) where they are activated by collision with a neutral gas. The activation causes some of the molecular ions to break up and the resulting fragment ions provide evidence of the original molecular structure. The spectrum of fragment ions is mass analysed in the second mass spectrometer, MS(2). Page 592 B ac k t o B a sic s Peptides and Proteins Analysis by MS Dynamic FAB Another development arising from FAB has been its transformation from a static to a dynamic technique, by allowing a continuous flow of a solution to travel from a reservoir through a capillary to the probe tip. Samples are injected either directly, or through a liquid chromatography (LC) column. The technique is known as Dynamic or Continuous Flow FAB/LSIMS and provides a convenient direct LC-MS coupling for the on-line analysis of mixtures, (Figure 2). Mixtures, as with the acyl coenzyme A factors shown in Figure 2, can be separated and analysed on-line. In peptide and protein work, the peptidic substance is often reacted (digested) with enzymes which cleave the peptide or protein at places along its backbone to give smaller peptides. This digest (mixture of peptides) must be separated into its components and the newly-formed peptides identified so that the original structure can be deduced (this is called ‘mapping’ and is something like assembling a linear jigsaw). LC-FAB-MS is well-suited to the separation of such mixtures and the identification of components through their molecular masses. However, not only the molecular mass of a peptide is important. The actual sequence (order) of amino acid residues making up the peptide chain is also important and FAB, which gives predominantly molecular mass information and few structural pointers, must be supplemented by another technique, MS-MS or tandem MS. Mass SpectrometryMass Spectrometry (MS-MS) Typically, a sample is analysed by FAB-MS to obtain a relative molecular mass and then by FAB-MS-MS to achieve structural information by fragmenting the molecular ion and examining the fragment ions. This is achieved by passing the molecular ion from the first mass analyser into a collision cell (Figure 3). Here collision gas (e.g. argon) is used to fragment this ion. The fragment ions produced are analysed by a second mass spectrometer. Mic rom as s U K Limite d Page 593 B ac k t o B a sic s Peptides and Proteins Analysis by MS 100% 90 80 70 60 50 40 30 20 10 0 m/z 300 915 H-Arg-Val-Tyr le-His-Pro-Phe-OH 669 532 784 400 mass 400 500 600 700 800 900 NH3 Phe Phe-Pro-H2 O Phe-Pro-His-H2O Arg-Val-Tyr-Ile Figure 4 Mic rom as s U K Limite d A typical FAB-MS-MS experiment on a peptide. The FAB-MS spectrum of angiotensin III is dominated by the protonated molecular ion [M+H]+ at m/z 932, confirming the molecular mass of 931. In an MS-MS experiment only the ion at m/z 932 was allowed to pass through the first mass analyser into the collision cell (Figure 3). On passing through the collision cell several fragment ions were produced and were all analysed by the second mass analyser to produce the spectrum shown here. The ion at m/z 915 arises by loss of NH3 from 932. The ions at m/z 784, 669, 532 and 400 arise respectively by loss of a phenylalanine residue; phenylalanine, proline and a water molecule; phenylalanine, proline, histidine and a water molecule; and arginine, valine, tyrosine and isoleucine, from 932. These fragments verify the expected sequence of amino acid residues in Angiotensin III. Page 594 B ac k t o B a sic s Peptides and Proteins Analysis by MS The fragments can only arise directly from the molecular ion and so provide useful sequence information for peptides. Peptides have been found to fragment by predictable pathways along their backbone from the C-terminus and/or the N-terminus, as shown by the example of angiotensin III (Figure 4). Tandem mass spectrometers most commonly used for MS-MS studies include the following analyser combinations, although many others are possible: 1. quadrupole - collision cell - quadrupole 2. magnetic/electrostatic analyser - collision cell - quadrupole 3. magnetic/electrostatic analyser - collision cell - magnetic/electrostatic analyser The collision cell used with the first two types (1, 2) is an RF-only quadrupole or a hexapole. This type of cell adds only a relatively small amount of energy to an ion during its collision with the cell gas. The third type uses a high energy collision cell which has the advantage of producing fragmentation of amino acid side chains as well as the ‘backbone’ fragmentation shown in Figure 4. This extra fragmentation gives information which permits differentiation of the two isomeric amino acid residues, leucine and isoleucine. Sequence information has been obtained by MS-MS on samples up to 2500 Da, which covers most enzyme digest generated peptides, often at low pmole levels, and usually in just a few minutes. Other Ion Sources Mic rom as s U K Limite d The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of this, the sensitivity of FAB is quite low and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. Page 595 B ac k t o B a sic s 100 Peptides and Proteins Analysis by MS 893.2 (a) 942.7 (n+1) + m1 998.1 1060.5 1131.1 848.6 n+ m2 1211.8 1304.9 808.2 1413.6 0 m/z 100 0 m/z 700 800 900 1000 1100 1200 1300 1400 1500 1600 16951.1 (c) 16200 16400 16600 16800 17000 17200 17400 17600 17800 (b) Mass to Charge Ratio (m/z) 1542.04 1413.59 1304.93 1211.80 1131.12 1060.46 998.11 942.75 893.15 848.57 808.21 771.49 No. of charges (n) 11 12 13 14 15 16 17 18 19 20 21 22 Mean S.D. Molecular Mass (RMM) 16951.40 16950.95 16950.94 16951.11 16951.62 16951.26 16950.67 16951.30 16950.71 16951.25 16951.14 16950.72 16951.09 ±0.30 Figure 5 A sample of the protein, horse heart myoglobin, was dissolved in acidified aqueous acetonitrile (1% formic acid in H2O/CH3CN, 1:1 v/v) at a concentration of 20 pmol/l. This sample was injected into a flow of the same solvent passing at 5 µl/min. into the electrospray source to give the mass spectrum of protonated molecular ions [M+nH]+ shown in (a). The measured m/z values are given in the table (b), along with the number of protons (charges; n) associated with each. The mean relative molecular mass (RMM) is 16951.09 ± 0.3 Da. Finally, the transformed spectrum, corresponding to the true relative molecular mass is shown in (c); the observed value is close to that calculated (16951.4), an error of only 0.002%. Mic rom as s U K Limite d Page 596 B ac k t o B a sic s Laser Desorption Mass Spectrometry (LDMS) Peptides and Proteins Analysis by MS This technique depends on the use of solid matrices (e.g., cinnamic acid derivatives) to absorb energy from a laser pulse so as to volatilize and ionize proteins premixed with the matrix. Mass analysis is achieved by a Time-of-Flight analyser which, as the name suggests, measures precisely the time taken for the ions to travel from the source through the flight tube to the detector. The heavier the ion, the longer the flight time. The spectrum generally contains a protonated [M+H+] or deprotonated [M-H]- molecular ion cluster, together with a doubly-charged and perhaps further multiply charged ions. Fragmentation, and hence sequence information, is usually absent. In principle there is no upper limit to the mass range and proteins as large as 200 kDa have been measured using as little as 1 pmole of material, making this one of the most sensitive techniques available. However, the resolution of this technique is low compared with other mass spectrometric methods, and the ions constituting the molecular mass cluster are unresolved. Heterogeneous proteins can present a problem, as the mass accuracy of 0.1% (e.g. 50 Da at 50 kDa) means that some post-translational modifications go undetected and mass changes associated with a single amino acid substitution would be unnoticed. Electrospray (ES) This second development has radically increased the use of mass spectrometry in biotechnology by providing an ionization technique capable of analysing large polar, thermally-sensitive biomolecules with unprecedented mass accuracy and good sensitivity. In ES, the sample, in solution, is passed through a narrow capillary and reaches an atmospheric pressure ionization source as a liquid spray. The voltage at the end of the capillary is significantly higher (3 kV) than that of the mass analyser and so the sample emerging is dispersed into an aerosol of highly charged droplets known as the electrospray. Evaporation of solvent, aided by a stream of nitrogen, causes a decrease in the size of the droplets until eventually multiply-charged ions from individual protein molecules, free from solvent, are released and can be mass analysed. Mic rom as s U K Limite d Page 597 B ac k t o B a sic s Peptides and Proteins Analysis by MS (a) 13+ etc. α13 = ( α globin + 13H) γ 13 = ( γ globin + 13H) 13+ etc. α15 α14 γ 16 γ 15 γ14 α13 γ13 β's α12 β's 1000 β's β 's 1100 1200 1300 (b) Normal α (15126.4) Gγ (15995.3) Normal β (15867.2) Aγ (16009.3) Sickle β (15837. 2) 0 mass Figure 6 Mic rom as s U K Limite d 15200 15400 15600 16000 16200 (a) Part of an electrospray spectrum of globins from the cord blood of a sickle cell carrier baby and, (b) the same after computer transformation onto a true molecular mass scale. Normal α, β and γ globins are clearly visible, along with the sickle cell variant, β. The two γ-globins (A, G), although having a mass difference of only 14 Da in 16,00 0Da, are easily distinguished from each other. Page 598 B ac k t o B a sic s Peptides and Proteins Analysis by MS The ions so produced are separated by their mass-to-charge (m/z) ratios. For peptides and proteins, the intact molecules become protonated with a number (n) of protons (H+). Thus, in place of the true molecular mass (M), molecular ions have a mass of [M+nH]. More importantly, the ion has n positive charges resulting from addition of the n protons (H+). Since the mass spectrometer does not measure mass directly but, rather, mass-to-charge (m/z) ratio the measured m/z value is [M+nH]/n. This last value is less than the true molecular mass, depending on the value of n. If the ion of true mass 20,000 Da carries 10 protons, for example, then the m/z value measured would be (20,000+10)/10 = 2001. This last m/z value is easy to measure accurately and, if its relationship to the true mass is known (n = 10), this means the true mass can be measured very accurately also. The multiply-charged ions have typical m/z values of <3000 Da, which means that conventional quadrupole or magnetic sector analysers can be used for mass measurement. Actually, the spectrum consists of a series of multiply-charged protonated molecular ions [M+nH]n+ for each component present in the sample. Each ion in the series differs by plus and minus one charge from adjacent ions ([M+nH]n+; n = an integer series for example, 1,2,3.... etc). Mathematical transformation of the spectrum produces a true molecular mass profile of the sample (Figure 5). Mic rom as s U K Limite d Page 599 B ac k t o B a sic s (a) 100 Peptides and Proteins Analysis by MS Normal α CHILD β Montreal - Chori β Sickle (β 6:Glu 0 mass (b) 100 15000 15200 Normal α 15400 15600 15000 Figure 7 Mic rom as s U K Limite d IIe) Val 15800 16000 MOTHER Normal β 0 mass ( β 87:Thr 15200 15400 15600 15800 β Montreal - Chori 16000 Electrospray mass spectra of globins from the blood of (a), a child diagnosed as having the sickle cell anaemia trait and (b) its mother. As well as the usual β-globin sickle cell variant at m/z 15837.2, a new variant (β-Montreal-Chori) appears at m/z 15879.3 and is observed in both the child and the mother. Page 600 B ac k t o B a sic s Proteins of Large Molecular Mass Peptides and Proteins Analysis by MS Whilst electrospray can be and is used for molecules of all molecular masses, it has had an especially marked impact on the measurement of accurate molecular mass for proteins. Traditionally, direct molecular mass measurement on proteins has been difficult, with values being obtained which were accurate to only tens or even hundreds of Daltons. The advent of electrospray now means that molecular masses of 20,000 Da and upwards can be measured with unprecedented accuracy (Figure 6). This level of accuracy means that it is also possible to identify post-translational modifications of proteins (e.g. glycosylation, acetylation, methylation, hydroxylation, etc.) and to detect mass changes associated with substitution or deletion of a single amino acid. A typical electrospray analysis can be completed in 15 minutes with as little as 1 pmole of protein. An analysis of the globins from the cord blood of a baby (Figure 6) showed quite clearly that five globins were present, viz., the normal ones (α, β, Gγ and Aγ) and a sickle cell variant (β). The last one is easily revealed in the mass spectrum even at a level of only 4% in the blood analysed. Whilst this example shows that small differences in large masses are easily discerned by electrospray methodology, it should be noted that absolute accuracy of mass measurement is unprecedentedly high. The accuracy is sufficiently high that substitution of one amino acid by another can be detected with ease. Figure 7 illustrates this following the discovery of a new variant of β-globin, called Hb Montreal-Chori, in which a threonine residue has been substituted by isoleucine at position-87 in the β-chain. The substitution caused a mass change from 15837.2 Da to 15879.3 Da. Mic rom as s U K Limite d Page 601 B ac k t o B a sic s Peptides and Proteins Analysis by MS On-Line HPLC of Peptide Mix Angiotensin 11 Unknown peptide 1010 Melanocyte stimulating hormone 100 TIC Bradykinin Bombesin Total Ion Current Luteinising hormone releasing hormone Methionine enkephalin Oxytocin Time (min.) Min 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 Figure 8(a) A mixture of peptides was separated by LC and the eluent was passed into an electrospray source. The total ion current trace (a) reveals the individual component peptides, each of which was identified through its measured accurate mass, an illustration of which is shown in (b) for luteinising hormone releasing hormone. 357 (30.345) 591.7 100 M+2H Luteinising Hormone Releasing Hormone 592.4 0 m/z 300 568.3 329.6 464.7 363.3 509.4 400 500 599.3 742.5 602.6 709.3 808.2 600 700 800 956.2 1030.5 900 1000 M+H 1183.2 1133.1 1100 1200 Figure 8(b) Mic rom as s U K Limite d Page 602 B ac k t o B a sic s Peptides and Proteins Analysis by MS Analysis of Peptide Mixtures The electrospray source can be coupled directly to a liquid chromatographic column so that, as components of a mixture emerge from the column, they are passed through the source to give accurate mass data. As an example, a mixture of the peptides shown in Figure 8(a) was separated by LC and accurately mass analysed by ES. Figure 8(b) is the mass spectrum of one of these peptides (luteinising hormone releasing hormone) which gave an abundant ion, representing a doubly-protonated molecule [M+2H]+ at m/z 592.4 and therefore, a true relative molecular mass (M) of 1182.8. Conclusion Intact peptides and proteins can be examined by a variety of new techniques, including MS-MS, dynamic-FAB and electrospray. Large masses of tens of thousands of Daltons can be accurately measured with unprecedented accuracy by electrospray. Mic rom as s U K Limite d Page 603 B ac k t o B a sic s Peptides and Proteins Analysis by MS This pa ge is intentiona lly b lan k. Mic rom as s U K Limite d Page 604