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Mass spectrometry of proteins
trends in analytical chemistry
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vol. 12, no. 10, 1993
Mass spectrometry of proteins Peter Roepstorff Odense, Denmark Several of the present mass spectrometric techniques have sufficient mass range and sensitivity to be viable alternatives or valuable supplements to traditional methods in protein chemistry. The precision of the molecular masses determined by mass spectrometry allows highly specific protein and peptide characterization as well as identification and localization of post translational modifications. In this article the principles and practical performance of the key techniques are discussed and examples of applications given.
Introduction Mass spectrometry (MS) of proteins has in the last decade developed into an effective tool in protein chemistry. This development started in the early 1980’s when two MS methods, plasma desorption (PD) [l] and fast atom bombardment (FAB) [2] MS, were both used to determine the molecular mass of insulin [3,4]. Insulin is among the smallest proteins (M,-6000) and, as it later became clear, also one of the easiest to analyze by MS. Nevertheless, this achievement marked the beginning of the rapid progress in peptide and protein analysis by MS. Practical mass ranges up to 25 000 Da and sensitivities in the high to low picomole range were obtained for FAB- and PDMS. Soon this development also created an increasing demand for improved performance of the MS techniques in terms of sensitivity and need to induce fragmentation of the low internal energy ions created by these soft ionization methods. These considerations led directly to the commercialization of tandem double focusing mass spectrometers for peptide sequence determination [5] and the subsequent incorporation of multichannel array detection [6]. During the time frame of these developments, two additional revolutionary ionization techniques were discovered and were quickly shown to be ideally suited to extending the
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mass range accessible for studies of biopolymers by a factor of ten. These are matrix assisted laser desorption/ionization (MALDI) [‘7] and electrospray ionization (ESI) [8] MS. In the following an account of the principles of the four methods with special emphasis on their practical performance will be presented. The interplay between MS and conventional protein chemistry will be illustrated with a few case studies from research in our group.
The mass spectrometric
methods
The principal constituents of a mass spectrometer are an ion source in which ions of the analyte are formed, a mass analyzer which separates the ions according to their mass to charge ratio (m/z) and an ion detector. The most common configurations and the performance of the methods are listed in Table 1. PD and MALDZ are both desorption/ionization methods in which ions are formed from the solid state by impact of fast heavy ion.s or photons, respectively. They both produce a pulsed ion beam which ideally combines with the time-of-flight analyzer. This analyzer has the advantage of unlimited mass range, simplicity, and relatively low cost. Consequently, all commercially available PD and MALDI instruments are time-of-flight (TOF) mass spectrometers. The main difference between the two methods lies in sample preparation (see below), processes of desorption and ionization, and analytical performance (Table 1). PD-MS is a surface ionization technique in which the sample is deposited or adsorbed on an appropriate surface. Practically all peptide and protein applications use adsorption of the sample molecules to a thin layer of nitrocellulose deposited on an aluminized mylar support [9]. The use of nitrocellulose has a number of advantages. It is compatible with most solvents used in traditional protein chemistry. Consequently, sample preparation can be effected simply by depositing a drop of sample solution on the surface followed by drying. It allows removal of buffers, many denaturants and
0 1993 Elsevier
Science
Publishers
B.V. All rights reserved
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TABLE 1. Summary of the mass spectrometric
methods described
Method
Commonly used mass analyzer
Matrix/solvent
Ion formation
Sensitivity limit (pmol)
Practical mass range
Mass accuracy (%)
PD
Time-of-flight
Nitrocellulose
l-10
20 000
0.05-0.2
MALDI
Time-of-flight
Organic solid
0.001-l
>250 000
0.01-0.2
FAB
Sector (or quadrupole) Quadrupole (or sector)
Organic liquid
Heavy ion induced desorption/ionization Photon induced desorption/ionization Light atom or ion induced desorption/ionization Electrospray ionization
I-500
Cl0 000
0.01-0.2
0.1-5
>iOO 000
0.001-0.02
ESI
Aqueous solution
salts by a simple washing procedure because the peptide or protein remains adsorbed to the surface. The easy removal of salts is especially important because traces of alkali salts are nearly always present in biological samples and their presence reduces the quality of the mass spectra. The use of nitrocellulose strongly enhances the molecular ion stability and abundance compared to most other supports, resulting in improved mass accuracy and sensitivity. In addition, as most of the sample is unaffected after recording a PD-spectrum, it allows further analysis of the sample after in situ reactions or recovery of the sample from the nitrocellulose [lo]. The following summarizes the specific features of PD-MS for analysis of proteins and peptides (for extensive reviews on the subject see, e.g., ref. 11). A sensitivity in the low picomole range and mass accuracy of 0.1% or better (Table 1) are both adequate for most practical protein studies. Although proteins beyond 20 kDa have been successfully analyzed by PD-MS, some much smaller proteins and certain modified proteins may fail. In general, we do not consider PD-MS universally applicable for proteins beyond 10 kDa and rarely for glycoproteins/peptides. Mixture analysis is possible but a certain degree of compound specific suppression may occur. The PD-mass spectrometer is very simple to operate, requires no tuning and can be automated. Mass calibration is simple and the spectra are easy to interpret. Sample preparation is straightforward and in situ purification and/or reactions as well as sample recovery is possible. Consequently, PD-MS is our preferred method for routine analyses of peptides up to 5 to 8 kDa, including peptides derived by enzymatic or chemical cleavage of proteins. MALDZ is a bulk process in which the analyte
molecules are mixed with a large molar excess of an appropriate matrix molecule to form a “solid solution” (for a recent review see ref. 12). The matrix molecules must exhibit a strong absorption at the wavelength of the laser light and, at least for protein analysis, the presence of a carboxylic acid group seems important. The most commonly used laser is a nitrogen laser with a wavelength of 337 nm and the corresponding matrices used for protein studies are, for example, dihydroxybenzoic acid and a variety of cinnamic acid derivatives. The sample is prepared by mixing a small volume of protein solution with a similar volume of saturated matrix solution, followed by drying of the solution on the probe. This seems quite simple, but recent investigations demonstrate that the best results are obtained when the protein is included in the matrix crystals [ 131 with concomitant exclusion of salts or other contaminants. The conditions for obtaining the best crystallization may vary between different proteins and certain areas of the surface may be better than others so that optimal areas must be searched for when acquiring spectra. These restraints put high requirements on the experience of the operator. The properties of MALDI are summarized below (for an extensive review see, e.g. ref. 14). Proteins with molecular masses well beyond 200 000 Da have been successfully analyzed by MALDI and the practical mass range seems to be unlimited. The present limitation is probably the ability of the detector to react on the very large and consequently rather slow ions. MALDI also seems to be universal so that all proteins, including heavily glycosylated proteins, can be analyzed. It is excellent for analysis of complex mixtures of peptides as well as proteins. The main limitation for the complexity of the mixture is the limited resolution of the TOF-ana-
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lyzer which does not allow resolution of components close in molecular mass. This, for example, becomes a problem in analysis of high-molecularmass glycoproteins where the components caused by heterogeneity in the carbohydrate chains cannot be resolved. MALDI is very tolerant towards the presence of salts or other contaminants present in the sample. The sensitivity in the low to mid femtomole range is the best of the described methods. Mass accuracy depends on the size of the protein and varies between 0.01 and 0.1% for small, respectively large proteins. The mass accuracy also depends on the laser irradiance and to obtain good results it is important to operate with irradiance close to the threshold for desorption. The calibration of the mass scale and hence the precision of the Mr measurement is influenced by several parameters including the laser n-radiance. Therefore, internal calibration standards close to the analyte in molecular mass and type are required for optimal precision. Thus, sample preparation and instrument handling presently is not as simple as for PD-MS. However, it must be taken into account that MALDI is a much younger technique than PD-MS, and it certainly has the instrumental potential to be as simple. Due to the limitations described, MALDI has in our laboratory not gained the same level of routine for analysis of peptides as PD-MS. On the other hand, MALDI is our first choice when ultimate sensitivity is required, for analysis of crude samples from early purification steps or even sometimes crude biological material, for glycoproteins and complex mixtures. FAB-MS is a desorption/ionization technique like the two previously described methods. The analyte molecules are dissolved in an appropriate liquid matrix (e.g., glycerol or thioglycerol). The desorption is effected by bombarding the surface of this matrix with atoms or ions in the keV energy range (in the latter case often termed liquid secondary ion mass spectrometry, LSI-MS). Accessories for FAB can be supplied with most sector and quadrupole instruments, resulting in widespread accessibility to the method. The practical mass range of approximately 10 000 Da brings most peptides and some small proteins in the scope of FAB-MS. The sensitivity strongly depends on the type and properties of the peptide/protein and is also affected by the solution environment, e.g., choice of liquid matrix, additives to enhance ionization, and presence of other components. In mix-
415
ture analysis pronounced suppression of components with low surface activity is often observed. Of particular value is the compatibility with MSMS which in combination with collision-induced dissociation (CID) of the “cold” MH+-ions allows direct MS sequencing of peptides up to about 3 kDa. High energy CID in sector instruments further allows a distinction between the isomeric amino acid residues Leu-Ile and the isobaric residues Gln-Lys due to side-chain fragmentation. (For an extensive review on FAB-MS and FABMS-MS of peptides see, e.g. ref. 15). Due to the complications imposed by the liquid matrix and the instrumental complexity, FAB-MS is rarely used in the daily routine of our protein studies. In ESZ-MS the protein sample is introduced in aqueous solution through a needle at high voltage. Ionization is effected by desolvation of droplets created under atmospheric pressure in an electrospray process between the needle and a grounded nozzle. This process, through a mechanism which at present is not quite understood, leads to a continuous production of a series of highly charged (multiprotonated) molecular ions which are channeled into the high vacuum of the mass spectrometer through a capillary or a series of differentially pumped skimmers. The continuous ion current and the highly charged state allow the use of scanning mass analyzers with a limited mass range. In effect most electrospray instruments used for protein analysis are quadrupole mass spectrometers with a mass range of 2000 to 4000. The ions exhibit very little fragmentation, but due to the coulombic repulsion which might be caused by the highly charged state, they fragment readily by a slight addition of energy. Thus, collision-induced dissociation can be performed either in the interface between the electrospray chamber and the mass spectrometer or in the second quadrupole of a triple quadrupole instrument. For proteins, this may lead to partial and, for peptides, to partial or complete series of sequence ions. However, to conclude that fragmentation of intact proteins is a practical way of partial sequencing of a protein is too preliminary at this point in time. ESI-MS is ideal for on-line coupling with separation techniques such as liquid chromatography or capillary electrophoresis because ionization takes place directly from liquid solutions. (for an extensive review on ESI-MS of proteins see, e.g. ref. 16). The specific features of ESI-MS of importance
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for protein analysis can be summarized as follows: The series of highly charged state molecular ions limits the mass range needed, bringing analysis of proteins well beyond 100 kDa, within the range of conventional mass analyzers. The mass accuracy gets very good (0.01% or better) because the molecular mass can be determined as the average of that determined for a number of peaks. Mass calibration can be performed with external standards as in conventional MS. Sample preparation is easy because it only requires dissolution of the protein in a concentration of the order of 0.1 pg/pl in a few l_tlof an appropriate solvent (e.g. water-methanolacetic acid, 10: 10: 1 v/v/v). Unfortunately, involatile buffers and salts cannot be accepted or only in very low concentrations, because their presence rapidly causes deposits on the surfaces, resulting in ion defocusing due to surface charging. Owing to its good dynamical range and resolution, ESIMS is well suited for analysis of mixtures and in particular to observe the presence of minor components. It is the method of choice for on-line coupling with separation techniques such as liquid chromatography or capillary electrophoresis and the value of LC-ESI-MS for mapping of enzymatic digests of proteins including the use of collision-induced dissociation for identification of phosphorylated and glycosylated peptides, has been demonstrated on several occasions [ 17-l 91. Instrumentally, ESI-MS is more complex than PD and MALDI and interpretation of the data for unknown samples, although mostly straightforward, may sometimes be complicated. In the author’s laboratory, ESI-MS is the preferred technique for precise molecular mass determination of purified proteins and for localization of minor components in protein preparations.
Examples of applications Characterization
of recombinant
amylases [20]
Barley a-amylase I expressed in yeast exhibited four bands of approximately equal intensity in isoelectric focusing. The proteins corresponding to the four bands were isolated, assayed for biological activity and their molecular mass determined by ESI-MS (Fig. 1). Two of the amylase forms for which the molecular masses were determined to 45 774 and 45 529 (AiVr 245) were fully biologically active, whereas the two other forms with a
100%
90%
I
80% 4
3t5+
35f I
LOO%-
b
A
r-
m-p-1 A
Fig. 1. ESI-spectrum of recombinant amylase 1 .l. (a) Complete spectrum. The inset shows the four majorforms identified. The shaded forms are biologically active whereas the glutathionylated forms (SG) are inactive. (b) Close view on the region corresponding to 35+ and 36+ charges. Component Ais glutathionylated but lack the C-terminal Thr-Arg. Components B, C, E and F are further C-terminal processed products and D is the non-glycosylated form.
molecular mass respectively 305 and 304 Da higher in mass exhibited a reduced activity. The mass difference between the two active forms corresponded to an expected C-terminal truncation by removal of the last two C-terminal residues (according to the cDNA-sequence Arg-Ser, A&l, is calculated at 243). The mass difference between these and the higher molecular mass inactive forms
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might correspond to glutathionylation of a cysteinyl residue (hM, is calculated at 305). To verify this hypothesis the high-molecular-mass forms of the active and inactive amylases were reduced with dithiothreitol (DTT) and analyzed by ESI-MS followed by alkylation with 2-vinyl-pyridine and MS analysis. As expected, the determined molecular mass showed a mass loss by treatment with DTT corresponding to loss of glutathione for the inactive form and no significant mass loss for the active form. Upon alkylation both forms showed a molecular mass increase corresponding to alkylation of four cysteinyl residues as expected from the cDNA-sequence. To identify the site of glutathionylation, protein engineered amylase genes were expressed each with one of the cysteines replaced by alanine and all with the C-terminal Arg-Ser codons removed. Activity assays and MS analysis identified Cys95 as the site of attachment of glutathione. MS analysis led to additional unexpected findings. The molecular masses of all components were approximately 350 above that calculated based on the cDNA sequence. However, a minor component 320 to 330 Da below was present in all spectra (Fig. 1) suggesting that the major products were 0-glycosylated with two hexose residues. The presence of glycosylation was subsequently confirmed by carbohydrate analysis of the recombinant product. The remaining molecular mass difference of 20 to 30 Da may be due either to an error in the cDNA sequence or to other modifications of the protein. Direct mapping by MALDIMS of the mixtures obtained by cleavage with trypsin and cyanogen bromide located the sequence error and the glycosylation site [21]. Minor components in the spectra (Fig. 1) also showed the presence of further C-terminal processing. This example clearly demonstrates the power of MS analysis in combination with appropriate protein chemical and genetic experiments for detection of post translational modifications in medium sized proteins. Based on the molecular masses determined, the presence of the following modifications were observed: Removal of the C-terminal Arg and Ser residues, glutathionylation of Cys95, glycosylation, and slight amounts of additional C-terminal truncation. Furthermore, the molecular masses determined were indicative of either a DNA sequence error or an additional modification, of which the former possibility could be confirmed by MS peptide mapping.
Search
forextended
parva/bumins
Parvalbumins are relatively low-molecularmass (cu. 11 kDa) Ca2+ or Mg2+-binding proteins which according to sequence studies constitute a subfamily of the large superfamily of calciumbinding proteins including, for example, troponinC and calmodulin. They are very abundant in muscles of lower vertebrates and most species contain a number of different parvalhumins, dominated by a- and P-parvalbumins containing 109 and 108 amino acid residues, respectively. A few longer parvalbumins have been observed. These “extended” parvalbumins are important in understanding the evolutionary relatiorrship between parvalbumins and other members of the calciumbinding protein family. Precise molecular mass determination seemed to be a good criterion to locate extended parvalbumins because an investigation of parvalbumins with known sequence showed a rather small spread in molecular masses, e.g. for seven different a-parvalbumins the mean molecular mass was 11 855 with 11756 and 12 125 being the extremes. Therefore, to search for extended parvalbumins and also to compare the performance of the MS techniques, we analyzed parvalbumin-containing fractions from different fish species by PD-MS, MALDI-MS and ESI-MS (Fig. 2) In total, ten fractions were analyzed as received without further purification. MALDI (with one exception) and ESI-MS immediately yielded the desired information, whereas removal of calcium ions and, for most fractions, further purification was necessary prior to PD-MS [22]. MALDl was the most sensitive and ESI-MS the most accurate of the methods. ESI-MS had the further advantage that minor components were readily identified in the spectra (Fig. 2~). The results of the MS analyses of the parvalbumins had several practical implications. They confirmed the sequence of a number of the parvalbumins, including the presence of a presumed but never really proven N-terminal acetylation in all but one of the parvalbumins (that of leopard shark). For one of the parvalbumins (pike, major component) the reported sequence was not in agreement with the molecular mass as measured by MS. A mass difference of 71 Da indicated that an alanyl residue was missing. This was confirmed and its position located as N-terminal by MS peptide mapping [23]. The presence in the ESI-spectra
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A 815
A13+ Pi0
1
I
9A,2+7
4
Measured mw’s A 11953 5 B: 12011 0 c: 93510 A
MH+
I
Fig. 2. Plasma desorption (a), matrix-assisted laser desorption/ionization (b) and electrospray spectrum (c) of parvalbumin 1 from carp. The electrospray spectrum in addition to the major component Ashows the presence of two minor components B and C. Component C is also observed in the MALDI spectrum. The peak in this spectrum at 5808.6 is recombinant human insulin used as calibration standard.
of dimers of a number of the parvalbumins could be related to the presence of a free cysteinyl residue in these parvalbumins and thus yielded additional structural information. The identification of parvalbumins has previously been based on retention time in the purification procedures and determination of the isoelectric point. This procedure is rather unspecific and creates some confusion when parvalbumins isolated in different laboratories are compared. The molecular mass determined by MS gives a much more specific identification. Only one of the parvalbumins (component 1 from carp) had a sufficiently high molecular mass to be a
candidate for an elongated parvalbumin. Sequence determination of this parvalbumin will be described in the following. Sequence determination carp
of parvalbumin
1 from
In our laboratory, we have for several years used an approach combining MS and automatic Edman degradation for sequence determination of proteins. The strategy was originally based on PD-MS [24] but has recently been modified to include MALDI and ESI-MS [25] as shown in Fig. 3.
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Molecular weight determination
(ESMS or LDMS). \
\ /
I
I
by LDMS, HPLC + PDMS, or LC-ESMS
I
HPLC-separation and M determination
\
\
Time-course enzymatic digestion monitored
I
I
PDMS
\
_;
N-terminal
sequence determination
C-terminal sequence determination
Alignment of adjoining peptides based on M, information from time-course
enzymatic
digestion, if possible
Supplementary alignment and verification of results by MS of products from an alternative enzymatic digestion.
Fig. 3. Strategy for protein sequencing combining mass spectrometry and Edman degradation.
Parvalbumin 1 from carp was one of the first proteins we sequenced with this new strategy [26]. First, the fraction measured in the previous study was submitted to further purification by HPLC. As expected from the previously recorded ESI-spectrum, this fraction in addition to the major component contained some minor components. Upon ESI-MS of the collected fractions, the major component was identified and its molecular mass determined at 11 954.3 Da. Three minor components were also identified, two of which according to their mass spectra were identical with the minor components identified previously (Fig. 2~). The major component was submitted to automatic Edman degradation and amino acid analysis from which it was concluded that the protein was N-terminally blocked and that endoproteinase Glu-C appeared to be the best choice to generate appropriately sized subpeptides for subsequent automatic sequencing. A time-course digestion monitored by MS spectrometry confirmed that the choice of enzyme was right, but unfortunately no partial digestion which would allow alignment of the peptides, was observed. A preparative digest was performed and the peptides separated by HPLC and their purity and molecular masses determined by PD-MS (Fig. 4). The sum of their determined molecular masses
(12 062.6 -6 x H20 = 11 954.5) confirmed that they accounted for the complete protein. Peptide S 1 could be identified as the N-terminal acetylated peptide because its molecular mass was 42 Da above that inferred from its amino acid composition. The other peptides were sequenced by automatic Edman degradation. Comparison of the determined sequences with the molecular masses showed that three of them were completely sequenced whereas the sequence of some amino acid residues was missing for the other three. These peptides were subdigested with chymotrypsin (Fig. 5). Based on the molecular masses determined by PD-MS of the derived peptides it was possible to select the peptides containing the missing sequences for sequencing. Peptide Sl was subdigested with trypsin yielding two peptides of which one could be sequenced. The other which contained the blocked N-terminal had the amino acid composition Met, Ala, and Lys, and the sequence either AcMet-Ala-Lys or AcAla-Met-Lys (Lys being the C-terminal residue because the peptide was derived by digestion with trypsin). To solve this question the protein was cleaved with cyanogen bromide (specific cleavage after Met) and the resulting product analyzed by ESI-MS (Fig. 6). Due to incomplete cleavage the spectrum showed two components. Their mass difference 244.9 Da corresponded to loss of AcAla-Met (calculated difference 244.3 Da) showing the N-terminal sequence to be AcAla-Met-Lys. As mentioned above, the time-course digestion did not allow direct alignment of the peptides. Instead, this was done based on the molecular masses (determined by PD-MS) of the peptides which were derived by digestion of the protein with trypsin. These data also served to confirm the determined sequence. As demonstrated in this example, the inclusion of MS analysis in the sequencing strategy has several advantageous features: l it reduces the total sample need, the number of cycles on the sequenator, and the time use; l it facilitates design of a sequencing strategy; l it ensures identification of modified residues; l it allows sequencing of blocked peptides either as in this specific case, by a combined biochemical and mass spectrometrical approach or in general by MS-MS; l perhaps most importantly, it gives an independent confirmation of the determined sequence.
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Mw’s measured Sl: 988.8 s2: 2715.2 s3: 987.2 S4: 2352.4 S5: 2139.5 S6: 2035.3 s7: 844.2
(calculated) (988.2) (2715.9) (986.2) (2352.6) (2139.4) (2035.2) (X43.9)
/
Fig. 4. Chromatogram from separation of the peptides derived by endoproteinase-Glu-C digestion of Parvalbumin 1 from Carp. The inset shows the PD-spectrum of peptide S7. The list contains the molecular masses of the peptides measured by PDMS and in parenthesis, the molecular masses calculated based on the later determined sequence.
Conclusion During the last decade the role of MS in protein chemistry has undergone rapid development from
CNBr
&
being a promising method through being a useful tool to being an indispensable technique. There are presently on1 r a few limitations in the mass range and types of proteins amenable to MS analysis. ‘,_,., ‘Ill2
f
”
T T
tT
Fig. 5. Schematics of the sequence of parvalbumin 1 from carp showing the V8 protease derived peptides. The shaded areas were sequenced by automatic Edman degradation. Sl is the N-terminally blocked peptide. The cleavage positions for subdigestion of S-peptides with chymotrypsin are indicated (C) and the positions for cleavage of the intact protein with cyanogen bromide (CNBr) and Trypsin (T).
Fig. 6. ESI-spectrum of cyanogen bromide cleaved parvalbumin 1 from carp.
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The sensitivity in the low picomole to femtomole range is as good as that of most other techniques in the field and adequate for most studies. The molecular mass is a unique property of a protein molecule which includes its amino acid content as well as post translational modifications The present mass accuracy permits protein characterization and identification with a certainty which is unsurpassed by any other technique, and it perfectly combines with the rapid progress in knowledge of genome structures.
References 1 D.F. Thorgersson, R.P. Skowronski and R.D. Macfarlane, Biochem. Biophys. Res. Commun., 60, (1974) 616. 2 M. Barber, R.S. Bordoli, R.D. Sedgwick and A.N. Tyler, J. Chem. Sot., Chem. Commun., (1981) 325.
3 P. Hakonsson, I. Karninsky, B.U.R. Sundqvist, J. Fohlman, J. Peterson, C. J. McNeal and R.D. Macfarlane, J. Am. Chem. Sot., 104 (1982) 2948. 4 A. Dell and H.R. Morris, Biochem. Biophys. Res. Commun., 106 (1982) 1456. 5 H.A. Scoble and K. Biemann, Science, 237 (1987) 992. 6 A.E. Ashcroft, R.A.C. Buchanan, G.J. Elliot, S. Evans, D.J. Milton and B. Wright, Proc. 36th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, 1988, p. 1156. 7 M. Karas and F. Hillenkamp, Anal. Chem., 66 (1988) 1084. 8 J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong and C.M. Whitehouse, Science, 246 (1989) 64. 9 G.l? Jonsson, A.B. Hedin, P.L. Hakansson, B.U.R. Sundqvist, G.S. Save, P.F. Nielsen, P. Roepstorff, K.E. Johansson, I. Kaminsky and M.S.L. Lindberg, Anal. Chem., 58 (1986) 1084. 10 S. Jespersen, G. Talbo and P. Roepstorff, Biol. Mass Spectrom., 22 (1993) 77. 11 R. Macfarlane, in D.M. Desiderio (Editor), Mass Spectrometry of Peptides, CRC-Press, Boca Raton, FL, 1990, p. 3 or P. Roepstorff, p. 65. 12 M. Karas and U. Bahr, TrendsAnal. Chem., 9 (1990) 321. 13 R. Beavis, Org. Mass Spectrom., 27 (1992) 864. 14 F. Hillenkamp and M. Karas, Methods Enzymol., 280 (1990) 193. 15 K. Biemann, Methods Enzymol., 193 (1990) 455. 16 R.D. Smith, J.A. Loo, M. Busman andH.R. Udseth, Mass Spec. Rev., 10 (1991) 359. 17 S.A. Carr, M.E. Hemling, M.F. Bean and G.D.
Roberts, Anal. Chem., 63 (1991) 2802. 18 V. Ling, A.W. Guzzetta, E. Canove-Davis, J.T. Stults, W.S. Hancock, T.R. Covey and B.I. Sushan, Anal. Chem., 63 (1991) 2909. 19 J.J. Conboy and J.D. Heinon, J. Am. Sot. Mass Spectrom., 3 (1992) 804. 20 M. Sogaard, J.S. Andersen, P. Roepstorff and B. Svensson, Biotechnology, in press. 21 J.S. Andersen, P. Roepstorff, M. Sogaard and B. Svensson, presented at the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, June, 1993, Abstract WD 76. 22 P. Roepstorff, K. Klarskov, J. Andersen, M. Mann, 0. Vorm, G. Etienne, and J. Parello, Znt. J. Mass Spectrom. Zen Proc., 111 (1991) 151. 23 K. Klarskov, Ph.D. Thesis, Odense University, 1991. 24 P. Roepstorff, K. Klarskov, and P. Hojrup, in B. Wittman-Liebold (Editor), Methods in Protein Sequence Analysis 1988, Springer Verlag, Berlin, 1989, p. 191. 25 P. Roepstorff and P. Hojrup, in K. Imahori and F. Sakyama (Editors), Methods in Protein Sequence Analysis 1992, Plenum Press, New York, 1993, p. 149. 26 P. Roepstorff, K. Rafn, G. Etienne and J. Parello, manuscript in preparation.
Dr. F! Roepstorff is at the Department of Molecular Biology, Odense University, DK-5230 Odense M, Denmark. -
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Mass spectrometry of proteins Peter Roepstorff Odense, Denmark Several of the present mass spectrometric techniques have sufficient mass range and sensitivity to be viable alternatives or valuable supplements to traditional methods in protein chemistry. The precision of the molecular masses determined by mass spectrometry allows highly specific protein and peptide characterization as well as identification and localization of post translational modifications. In this article the principles and practical performance of the key techniques are discussed and examples of applications given.
Introduction Mass spectrometry (MS) of proteins has in the last decade developed into an effective tool in protein chemistry. This development started in the early 1980’s when two MS methods, plasma desorption (PD) [l] and fast atom bombardment (FAB) [2] MS, were both used to determine the molecular mass of insulin [3,4]. Insulin is among the smallest proteins (M,-6000) and, as it later became clear, also one of the easiest to analyze by MS. Nevertheless, this achievement marked the beginning of the rapid progress in peptide and protein analysis by MS. Practical mass ranges up to 25 000 Da and sensitivities in the high to low picomole range were obtained for FAB- and PDMS. Soon this development also created an increasing demand for improved performance of the MS techniques in terms of sensitivity and need to induce fragmentation of the low internal energy ions created by these soft ionization methods. These considerations led directly to the commercialization of tandem double focusing mass spectrometers for peptide sequence determination [5] and the subsequent incorporation of multichannel array detection [6]. During the time frame of these developments, two additional revolutionary ionization techniques were discovered and were quickly shown to be ideally suited to extending the
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mass range accessible for studies of biopolymers by a factor of ten. These are matrix assisted laser desorption/ionization (MALDI) [‘7] and electrospray ionization (ESI) [8] MS. In the following an account of the principles of the four methods with special emphasis on their practical performance will be presented. The interplay between MS and conventional protein chemistry will be illustrated with a few case studies from research in our group.
The mass spectrometric
methods
The principal constituents of a mass spectrometer are an ion source in which ions of the analyte are formed, a mass analyzer which separates the ions according to their mass to charge ratio (m/z) and an ion detector. The most common configurations and the performance of the methods are listed in Table 1. PD and MALDZ are both desorption/ionization methods in which ions are formed from the solid state by impact of fast heavy ion.s or photons, respectively. They both produce a pulsed ion beam which ideally combines with the time-of-flight analyzer. This analyzer has the advantage of unlimited mass range, simplicity, and relatively low cost. Consequently, all commercially available PD and MALDI instruments are time-of-flight (TOF) mass spectrometers. The main difference between the two methods lies in sample preparation (see below), processes of desorption and ionization, and analytical performance (Table 1). PD-MS is a surface ionization technique in which the sample is deposited or adsorbed on an appropriate surface. Practically all peptide and protein applications use adsorption of the sample molecules to a thin layer of nitrocellulose deposited on an aluminized mylar support [9]. The use of nitrocellulose has a number of advantages. It is compatible with most solvents used in traditional protein chemistry. Consequently, sample preparation can be effected simply by depositing a drop of sample solution on the surface followed by drying. It allows removal of buffers, many denaturants and
0 1993 Elsevier
Science
Publishers
B.V. All rights reserved
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TABLE 1. Summary of the mass spectrometric
methods described
Method
Commonly used mass analyzer
Matrix/solvent
Ion formation
Sensitivity limit (pmol)
Practical mass range
Mass accuracy (%)
PD
Time-of-flight
Nitrocellulose
l-10
20 000
0.05-0.2
MALDI
Time-of-flight
Organic solid
0.001-l
>250 000
0.01-0.2
FAB
Sector (or quadrupole) Quadrupole (or sector)
Organic liquid
Heavy ion induced desorption/ionization Photon induced desorption/ionization Light atom or ion induced desorption/ionization Electrospray ionization
I-500
Cl0 000
0.01-0.2
0.1-5
>iOO 000
0.001-0.02
ESI
Aqueous solution
salts by a simple washing procedure because the peptide or protein remains adsorbed to the surface. The easy removal of salts is especially important because traces of alkali salts are nearly always present in biological samples and their presence reduces the quality of the mass spectra. The use of nitrocellulose strongly enhances the molecular ion stability and abundance compared to most other supports, resulting in improved mass accuracy and sensitivity. In addition, as most of the sample is unaffected after recording a PD-spectrum, it allows further analysis of the sample after in situ reactions or recovery of the sample from the nitrocellulose [lo]. The following summarizes the specific features of PD-MS for analysis of proteins and peptides (for extensive reviews on the subject see, e.g., ref. 11). A sensitivity in the low picomole range and mass accuracy of 0.1% or better (Table 1) are both adequate for most practical protein studies. Although proteins beyond 20 kDa have been successfully analyzed by PD-MS, some much smaller proteins and certain modified proteins may fail. In general, we do not consider PD-MS universally applicable for proteins beyond 10 kDa and rarely for glycoproteins/peptides. Mixture analysis is possible but a certain degree of compound specific suppression may occur. The PD-mass spectrometer is very simple to operate, requires no tuning and can be automated. Mass calibration is simple and the spectra are easy to interpret. Sample preparation is straightforward and in situ purification and/or reactions as well as sample recovery is possible. Consequently, PD-MS is our preferred method for routine analyses of peptides up to 5 to 8 kDa, including peptides derived by enzymatic or chemical cleavage of proteins. MALDZ is a bulk process in which the analyte
molecules are mixed with a large molar excess of an appropriate matrix molecule to form a “solid solution” (for a recent review see ref. 12). The matrix molecules must exhibit a strong absorption at the wavelength of the laser light and, at least for protein analysis, the presence of a carboxylic acid group seems important. The most commonly used laser is a nitrogen laser with a wavelength of 337 nm and the corresponding matrices used for protein studies are, for example, dihydroxybenzoic acid and a variety of cinnamic acid derivatives. The sample is prepared by mixing a small volume of protein solution with a similar volume of saturated matrix solution, followed by drying of the solution on the probe. This seems quite simple, but recent investigations demonstrate that the best results are obtained when the protein is included in the matrix crystals [ 131 with concomitant exclusion of salts or other contaminants. The conditions for obtaining the best crystallization may vary between different proteins and certain areas of the surface may be better than others so that optimal areas must be searched for when acquiring spectra. These restraints put high requirements on the experience of the operator. The properties of MALDI are summarized below (for an extensive review see, e.g. ref. 14). Proteins with molecular masses well beyond 200 000 Da have been successfully analyzed by MALDI and the practical mass range seems to be unlimited. The present limitation is probably the ability of the detector to react on the very large and consequently rather slow ions. MALDI also seems to be universal so that all proteins, including heavily glycosylated proteins, can be analyzed. It is excellent for analysis of complex mixtures of peptides as well as proteins. The main limitation for the complexity of the mixture is the limited resolution of the TOF-ana-
trends in analytical chemistry, vol. 12, no. 10, 1993
lyzer which does not allow resolution of components close in molecular mass. This, for example, becomes a problem in analysis of high-molecularmass glycoproteins where the components caused by heterogeneity in the carbohydrate chains cannot be resolved. MALDI is very tolerant towards the presence of salts or other contaminants present in the sample. The sensitivity in the low to mid femtomole range is the best of the described methods. Mass accuracy depends on the size of the protein and varies between 0.01 and 0.1% for small, respectively large proteins. The mass accuracy also depends on the laser irradiance and to obtain good results it is important to operate with irradiance close to the threshold for desorption. The calibration of the mass scale and hence the precision of the Mr measurement is influenced by several parameters including the laser n-radiance. Therefore, internal calibration standards close to the analyte in molecular mass and type are required for optimal precision. Thus, sample preparation and instrument handling presently is not as simple as for PD-MS. However, it must be taken into account that MALDI is a much younger technique than PD-MS, and it certainly has the instrumental potential to be as simple. Due to the limitations described, MALDI has in our laboratory not gained the same level of routine for analysis of peptides as PD-MS. On the other hand, MALDI is our first choice when ultimate sensitivity is required, for analysis of crude samples from early purification steps or even sometimes crude biological material, for glycoproteins and complex mixtures. FAB-MS is a desorption/ionization technique like the two previously described methods. The analyte molecules are dissolved in an appropriate liquid matrix (e.g., glycerol or thioglycerol). The desorption is effected by bombarding the surface of this matrix with atoms or ions in the keV energy range (in the latter case often termed liquid secondary ion mass spectrometry, LSI-MS). Accessories for FAB can be supplied with most sector and quadrupole instruments, resulting in widespread accessibility to the method. The practical mass range of approximately 10 000 Da brings most peptides and some small proteins in the scope of FAB-MS. The sensitivity strongly depends on the type and properties of the peptide/protein and is also affected by the solution environment, e.g., choice of liquid matrix, additives to enhance ionization, and presence of other components. In mix-
415
ture analysis pronounced suppression of components with low surface activity is often observed. Of particular value is the compatibility with MSMS which in combination with collision-induced dissociation (CID) of the “cold” MH+-ions allows direct MS sequencing of peptides up to about 3 kDa. High energy CID in sector instruments further allows a distinction between the isomeric amino acid residues Leu-Ile and the isobaric residues Gln-Lys due to side-chain fragmentation. (For an extensive review on FAB-MS and FABMS-MS of peptides see, e.g. ref. 15). Due to the complications imposed by the liquid matrix and the instrumental complexity, FAB-MS is rarely used in the daily routine of our protein studies. In ESZ-MS the protein sample is introduced in aqueous solution through a needle at high voltage. Ionization is effected by desolvation of droplets created under atmospheric pressure in an electrospray process between the needle and a grounded nozzle. This process, through a mechanism which at present is not quite understood, leads to a continuous production of a series of highly charged (multiprotonated) molecular ions which are channeled into the high vacuum of the mass spectrometer through a capillary or a series of differentially pumped skimmers. The continuous ion current and the highly charged state allow the use of scanning mass analyzers with a limited mass range. In effect most electrospray instruments used for protein analysis are quadrupole mass spectrometers with a mass range of 2000 to 4000. The ions exhibit very little fragmentation, but due to the coulombic repulsion which might be caused by the highly charged state, they fragment readily by a slight addition of energy. Thus, collision-induced dissociation can be performed either in the interface between the electrospray chamber and the mass spectrometer or in the second quadrupole of a triple quadrupole instrument. For proteins, this may lead to partial and, for peptides, to partial or complete series of sequence ions. However, to conclude that fragmentation of intact proteins is a practical way of partial sequencing of a protein is too preliminary at this point in time. ESI-MS is ideal for on-line coupling with separation techniques such as liquid chromatography or capillary electrophoresis because ionization takes place directly from liquid solutions. (for an extensive review on ESI-MS of proteins see, e.g. ref. 16). The specific features of ESI-MS of importance
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for protein analysis can be summarized as follows: The series of highly charged state molecular ions limits the mass range needed, bringing analysis of proteins well beyond 100 kDa, within the range of conventional mass analyzers. The mass accuracy gets very good (0.01% or better) because the molecular mass can be determined as the average of that determined for a number of peaks. Mass calibration can be performed with external standards as in conventional MS. Sample preparation is easy because it only requires dissolution of the protein in a concentration of the order of 0.1 pg/pl in a few l_tlof an appropriate solvent (e.g. water-methanolacetic acid, 10: 10: 1 v/v/v). Unfortunately, involatile buffers and salts cannot be accepted or only in very low concentrations, because their presence rapidly causes deposits on the surfaces, resulting in ion defocusing due to surface charging. Owing to its good dynamical range and resolution, ESIMS is well suited for analysis of mixtures and in particular to observe the presence of minor components. It is the method of choice for on-line coupling with separation techniques such as liquid chromatography or capillary electrophoresis and the value of LC-ESI-MS for mapping of enzymatic digests of proteins including the use of collision-induced dissociation for identification of phosphorylated and glycosylated peptides, has been demonstrated on several occasions [ 17-l 91. Instrumentally, ESI-MS is more complex than PD and MALDI and interpretation of the data for unknown samples, although mostly straightforward, may sometimes be complicated. In the author’s laboratory, ESI-MS is the preferred technique for precise molecular mass determination of purified proteins and for localization of minor components in protein preparations.
Examples of applications Characterization
of recombinant
amylases [20]
Barley a-amylase I expressed in yeast exhibited four bands of approximately equal intensity in isoelectric focusing. The proteins corresponding to the four bands were isolated, assayed for biological activity and their molecular mass determined by ESI-MS (Fig. 1). Two of the amylase forms for which the molecular masses were determined to 45 774 and 45 529 (AiVr 245) were fully biologically active, whereas the two other forms with a
100%
90%
I
80% 4
3t5+
35f I
LOO%-
b
A
r-
m-p-1 A
Fig. 1. ESI-spectrum of recombinant amylase 1 .l. (a) Complete spectrum. The inset shows the four majorforms identified. The shaded forms are biologically active whereas the glutathionylated forms (SG) are inactive. (b) Close view on the region corresponding to 35+ and 36+ charges. Component Ais glutathionylated but lack the C-terminal Thr-Arg. Components B, C, E and F are further C-terminal processed products and D is the non-glycosylated form.
molecular mass respectively 305 and 304 Da higher in mass exhibited a reduced activity. The mass difference between the two active forms corresponded to an expected C-terminal truncation by removal of the last two C-terminal residues (according to the cDNA-sequence Arg-Ser, A&l, is calculated at 243). The mass difference between these and the higher molecular mass inactive forms
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might correspond to glutathionylation of a cysteinyl residue (hM, is calculated at 305). To verify this hypothesis the high-molecular-mass forms of the active and inactive amylases were reduced with dithiothreitol (DTT) and analyzed by ESI-MS followed by alkylation with 2-vinyl-pyridine and MS analysis. As expected, the determined molecular mass showed a mass loss by treatment with DTT corresponding to loss of glutathione for the inactive form and no significant mass loss for the active form. Upon alkylation both forms showed a molecular mass increase corresponding to alkylation of four cysteinyl residues as expected from the cDNA-sequence. To identify the site of glutathionylation, protein engineered amylase genes were expressed each with one of the cysteines replaced by alanine and all with the C-terminal Arg-Ser codons removed. Activity assays and MS analysis identified Cys95 as the site of attachment of glutathione. MS analysis led to additional unexpected findings. The molecular masses of all components were approximately 350 above that calculated based on the cDNA sequence. However, a minor component 320 to 330 Da below was present in all spectra (Fig. 1) suggesting that the major products were 0-glycosylated with two hexose residues. The presence of glycosylation was subsequently confirmed by carbohydrate analysis of the recombinant product. The remaining molecular mass difference of 20 to 30 Da may be due either to an error in the cDNA sequence or to other modifications of the protein. Direct mapping by MALDIMS of the mixtures obtained by cleavage with trypsin and cyanogen bromide located the sequence error and the glycosylation site [21]. Minor components in the spectra (Fig. 1) also showed the presence of further C-terminal processing. This example clearly demonstrates the power of MS analysis in combination with appropriate protein chemical and genetic experiments for detection of post translational modifications in medium sized proteins. Based on the molecular masses determined, the presence of the following modifications were observed: Removal of the C-terminal Arg and Ser residues, glutathionylation of Cys95, glycosylation, and slight amounts of additional C-terminal truncation. Furthermore, the molecular masses determined were indicative of either a DNA sequence error or an additional modification, of which the former possibility could be confirmed by MS peptide mapping.
Search
forextended
parva/bumins
Parvalbumins are relatively low-molecularmass (cu. 11 kDa) Ca2+ or Mg2+-binding proteins which according to sequence studies constitute a subfamily of the large superfamily of calciumbinding proteins including, for example, troponinC and calmodulin. They are very abundant in muscles of lower vertebrates and most species contain a number of different parvalhumins, dominated by a- and P-parvalbumins containing 109 and 108 amino acid residues, respectively. A few longer parvalbumins have been observed. These “extended” parvalbumins are important in understanding the evolutionary relatiorrship between parvalbumins and other members of the calciumbinding protein family. Precise molecular mass determination seemed to be a good criterion to locate extended parvalbumins because an investigation of parvalbumins with known sequence showed a rather small spread in molecular masses, e.g. for seven different a-parvalbumins the mean molecular mass was 11 855 with 11756 and 12 125 being the extremes. Therefore, to search for extended parvalbumins and also to compare the performance of the MS techniques, we analyzed parvalbumin-containing fractions from different fish species by PD-MS, MALDI-MS and ESI-MS (Fig. 2) In total, ten fractions were analyzed as received without further purification. MALDI (with one exception) and ESI-MS immediately yielded the desired information, whereas removal of calcium ions and, for most fractions, further purification was necessary prior to PD-MS [22]. MALDl was the most sensitive and ESI-MS the most accurate of the methods. ESI-MS had the further advantage that minor components were readily identified in the spectra (Fig. 2~). The results of the MS analyses of the parvalbumins had several practical implications. They confirmed the sequence of a number of the parvalbumins, including the presence of a presumed but never really proven N-terminal acetylation in all but one of the parvalbumins (that of leopard shark). For one of the parvalbumins (pike, major component) the reported sequence was not in agreement with the molecular mass as measured by MS. A mass difference of 71 Da indicated that an alanyl residue was missing. This was confirmed and its position located as N-terminal by MS peptide mapping [23]. The presence in the ESI-spectra
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A 815
A13+ Pi0
1
I
9A,2+7
4
Measured mw’s A 11953 5 B: 12011 0 c: 93510 A
MH+
I
Fig. 2. Plasma desorption (a), matrix-assisted laser desorption/ionization (b) and electrospray spectrum (c) of parvalbumin 1 from carp. The electrospray spectrum in addition to the major component Ashows the presence of two minor components B and C. Component C is also observed in the MALDI spectrum. The peak in this spectrum at 5808.6 is recombinant human insulin used as calibration standard.
of dimers of a number of the parvalbumins could be related to the presence of a free cysteinyl residue in these parvalbumins and thus yielded additional structural information. The identification of parvalbumins has previously been based on retention time in the purification procedures and determination of the isoelectric point. This procedure is rather unspecific and creates some confusion when parvalbumins isolated in different laboratories are compared. The molecular mass determined by MS gives a much more specific identification. Only one of the parvalbumins (component 1 from carp) had a sufficiently high molecular mass to be a
candidate for an elongated parvalbumin. Sequence determination of this parvalbumin will be described in the following. Sequence determination carp
of parvalbumin
1 from
In our laboratory, we have for several years used an approach combining MS and automatic Edman degradation for sequence determination of proteins. The strategy was originally based on PD-MS [24] but has recently been modified to include MALDI and ESI-MS [25] as shown in Fig. 3.
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Molecular weight determination
(ESMS or LDMS). \
\ /
I
I
by LDMS, HPLC + PDMS, or LC-ESMS
I
HPLC-separation and M determination
\
\
Time-course enzymatic digestion monitored
I
I
PDMS
\
_;
N-terminal
sequence determination
C-terminal sequence determination
Alignment of adjoining peptides based on M, information from time-course
enzymatic
digestion, if possible
Supplementary alignment and verification of results by MS of products from an alternative enzymatic digestion.
Fig. 3. Strategy for protein sequencing combining mass spectrometry and Edman degradation.
Parvalbumin 1 from carp was one of the first proteins we sequenced with this new strategy [26]. First, the fraction measured in the previous study was submitted to further purification by HPLC. As expected from the previously recorded ESI-spectrum, this fraction in addition to the major component contained some minor components. Upon ESI-MS of the collected fractions, the major component was identified and its molecular mass determined at 11 954.3 Da. Three minor components were also identified, two of which according to their mass spectra were identical with the minor components identified previously (Fig. 2~). The major component was submitted to automatic Edman degradation and amino acid analysis from which it was concluded that the protein was N-terminally blocked and that endoproteinase Glu-C appeared to be the best choice to generate appropriately sized subpeptides for subsequent automatic sequencing. A time-course digestion monitored by MS spectrometry confirmed that the choice of enzyme was right, but unfortunately no partial digestion which would allow alignment of the peptides, was observed. A preparative digest was performed and the peptides separated by HPLC and their purity and molecular masses determined by PD-MS (Fig. 4). The sum of their determined molecular masses
(12 062.6 -6 x H20 = 11 954.5) confirmed that they accounted for the complete protein. Peptide S 1 could be identified as the N-terminal acetylated peptide because its molecular mass was 42 Da above that inferred from its amino acid composition. The other peptides were sequenced by automatic Edman degradation. Comparison of the determined sequences with the molecular masses showed that three of them were completely sequenced whereas the sequence of some amino acid residues was missing for the other three. These peptides were subdigested with chymotrypsin (Fig. 5). Based on the molecular masses determined by PD-MS of the derived peptides it was possible to select the peptides containing the missing sequences for sequencing. Peptide Sl was subdigested with trypsin yielding two peptides of which one could be sequenced. The other which contained the blocked N-terminal had the amino acid composition Met, Ala, and Lys, and the sequence either AcMet-Ala-Lys or AcAla-Met-Lys (Lys being the C-terminal residue because the peptide was derived by digestion with trypsin). To solve this question the protein was cleaved with cyanogen bromide (specific cleavage after Met) and the resulting product analyzed by ESI-MS (Fig. 6). Due to incomplete cleavage the spectrum showed two components. Their mass difference 244.9 Da corresponded to loss of AcAla-Met (calculated difference 244.3 Da) showing the N-terminal sequence to be AcAla-Met-Lys. As mentioned above, the time-course digestion did not allow direct alignment of the peptides. Instead, this was done based on the molecular masses (determined by PD-MS) of the peptides which were derived by digestion of the protein with trypsin. These data also served to confirm the determined sequence. As demonstrated in this example, the inclusion of MS analysis in the sequencing strategy has several advantageous features: l it reduces the total sample need, the number of cycles on the sequenator, and the time use; l it facilitates design of a sequencing strategy; l it ensures identification of modified residues; l it allows sequencing of blocked peptides either as in this specific case, by a combined biochemical and mass spectrometrical approach or in general by MS-MS; l perhaps most importantly, it gives an independent confirmation of the determined sequence.
trends in analytical chemistry, vol. 12, no. 10. 1993
Mw’s measured Sl: 988.8 s2: 2715.2 s3: 987.2 S4: 2352.4 S5: 2139.5 S6: 2035.3 s7: 844.2
(calculated) (988.2) (2715.9) (986.2) (2352.6) (2139.4) (2035.2) (X43.9)
/
Fig. 4. Chromatogram from separation of the peptides derived by endoproteinase-Glu-C digestion of Parvalbumin 1 from Carp. The inset shows the PD-spectrum of peptide S7. The list contains the molecular masses of the peptides measured by PDMS and in parenthesis, the molecular masses calculated based on the later determined sequence.
Conclusion During the last decade the role of MS in protein chemistry has undergone rapid development from
CNBr
&
being a promising method through being a useful tool to being an indispensable technique. There are presently on1 r a few limitations in the mass range and types of proteins amenable to MS analysis. ‘,_,., ‘Ill2
f
”
T T
tT
Fig. 5. Schematics of the sequence of parvalbumin 1 from carp showing the V8 protease derived peptides. The shaded areas were sequenced by automatic Edman degradation. Sl is the N-terminally blocked peptide. The cleavage positions for subdigestion of S-peptides with chymotrypsin are indicated (C) and the positions for cleavage of the intact protein with cyanogen bromide (CNBr) and Trypsin (T).
Fig. 6. ESI-spectrum of cyanogen bromide cleaved parvalbumin 1 from carp.
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The sensitivity in the low picomole to femtomole range is as good as that of most other techniques in the field and adequate for most studies. The molecular mass is a unique property of a protein molecule which includes its amino acid content as well as post translational modifications The present mass accuracy permits protein characterization and identification with a certainty which is unsurpassed by any other technique, and it perfectly combines with the rapid progress in knowledge of genome structures.
References 1 D.F. Thorgersson, R.P. Skowronski and R.D. Macfarlane, Biochem. Biophys. Res. Commun., 60, (1974) 616. 2 M. Barber, R.S. Bordoli, R.D. Sedgwick and A.N. Tyler, J. Chem. Sot., Chem. Commun., (1981) 325.
3 P. Hakonsson, I. Karninsky, B.U.R. Sundqvist, J. Fohlman, J. Peterson, C. J. McNeal and R.D. Macfarlane, J. Am. Chem. Sot., 104 (1982) 2948. 4 A. Dell and H.R. Morris, Biochem. Biophys. Res. Commun., 106 (1982) 1456. 5 H.A. Scoble and K. Biemann, Science, 237 (1987) 992. 6 A.E. Ashcroft, R.A.C. Buchanan, G.J. Elliot, S. Evans, D.J. Milton and B. Wright, Proc. 36th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, 1988, p. 1156. 7 M. Karas and F. Hillenkamp, Anal. Chem., 66 (1988) 1084. 8 J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong and C.M. Whitehouse, Science, 246 (1989) 64. 9 G.l? Jonsson, A.B. Hedin, P.L. Hakansson, B.U.R. Sundqvist, G.S. Save, P.F. Nielsen, P. Roepstorff, K.E. Johansson, I. Kaminsky and M.S.L. Lindberg, Anal. Chem., 58 (1986) 1084. 10 S. Jespersen, G. Talbo and P. Roepstorff, Biol. Mass Spectrom., 22 (1993) 77. 11 R. Macfarlane, in D.M. Desiderio (Editor), Mass Spectrometry of Peptides, CRC-Press, Boca Raton, FL, 1990, p. 3 or P. Roepstorff, p. 65. 12 M. Karas and U. Bahr, TrendsAnal. Chem., 9 (1990) 321. 13 R. Beavis, Org. Mass Spectrom., 27 (1992) 864. 14 F. Hillenkamp and M. Karas, Methods Enzymol., 280 (1990) 193. 15 K. Biemann, Methods Enzymol., 193 (1990) 455. 16 R.D. Smith, J.A. Loo, M. Busman andH.R. Udseth, Mass Spec. Rev., 10 (1991) 359. 17 S.A. Carr, M.E. Hemling, M.F. Bean and G.D.
Roberts, Anal. Chem., 63 (1991) 2802. 18 V. Ling, A.W. Guzzetta, E. Canove-Davis, J.T. Stults, W.S. Hancock, T.R. Covey and B.I. Sushan, Anal. Chem., 63 (1991) 2909. 19 J.J. Conboy and J.D. Heinon, J. Am. Sot. Mass Spectrom., 3 (1992) 804. 20 M. Sogaard, J.S. Andersen, P. Roepstorff and B. Svensson, Biotechnology, in press. 21 J.S. Andersen, P. Roepstorff, M. Sogaard and B. Svensson, presented at the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, June, 1993, Abstract WD 76. 22 P. Roepstorff, K. Klarskov, J. Andersen, M. Mann, 0. Vorm, G. Etienne, and J. Parello, Znt. J. Mass Spectrom. Zen Proc., 111 (1991) 151. 23 K. Klarskov, Ph.D. Thesis, Odense University, 1991. 24 P. Roepstorff, K. Klarskov, and P. Hojrup, in B. Wittman-Liebold (Editor), Methods in Protein Sequence Analysis 1988, Springer Verlag, Berlin, 1989, p. 191. 25 P. Roepstorff and P. Hojrup, in K. Imahori and F. Sakyama (Editors), Methods in Protein Sequence Analysis 1992, Plenum Press, New York, 1993, p. 149. 26 P. Roepstorff, K. Rafn, G. Etienne and J. Parello, manuscript in preparation.
Dr. F! Roepstorff is at the Department of Molecular Biology, Odense University, DK-5230 Odense M, Denmark. -
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