New methods in mass spectrometry of proteins

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Chung,K-T.,Dickson,IS. and Cmuse,I.D.(1989)Appl. Environ. MicrobioL 55, 1329-1333 DeKlerk,H.C.and Stall,I.A.~19671I. Gen.Micmbiol. 48, 309-316 Upreti,G.C.and Hinsdill,R.D. (1975)Antimicrob. Agents Chemother. 7, 139-145

27 Joerger,M.C. and Klaenhammer,T.R. (1990)J. Bacteriol. 172, 6339-6347 Bare(oot,S.F.and Klaenhammer,T.R.(1984)Antimicro~. Agents Chemother. 26, 328-334 29 Muriana,P.M.and Klaenhammer,T.R.(19911Appl.Environ. Microbiol. 57, 114-121 30 Daeschel,M.A.,McKenny,M.C.and McDonald,t.C. (1986)Abstr. Ann. Meet. Am. Soc. MicrobioL 86, 277 31 Schillinger,U. and LOcke,F-K.(1989)Appl. Environ. MicrobioL 55, 28

32

1901-1906 Morlvedl, C.I., Nissen-Meyer,l., Sletten,K. and Nes,I.F. (1991) AppL Environ. Microbiol. 57, 1829-1834

33 34

Rammelsberg,M., Muller,E.and Radler,F. (1990)Arch. Microbiol. i 54, 249-252 tOcke,F-K.and Schillinger,U. (19901FEMSMicrobiol.Rev. 87,

Abstract E3 35 Morlvedl, C.I. and Nes, I.F. (I 9901I. Gen. Microbiol. 136, 1601-I 607 36 Schillinger,U. and Holzapfel, W.H. (I 990) Food Microbiol. 7, 305-310

37 Ahn,C. and Stiles,M.E. (1990)I. Appl. Bacteriol. 69, 302-310 311 Ahn,C. and Stiles, M.E. (1990) Appl. Environ. Microbiol. 56, 39

2503-2510 Harding,C.D.and Shaw, B.G.I19901J.Appl. BacterioL 69, 648-654

40 Hastings,l.W.and Stiles,M.E.(1991) I. AppL BacterioL 70, i 27-134 41 Orberg,P.K.and Sandine,W.E.(19841AppLEnviron. MicrobioL 48, 1129-1133 42 Shaw,B.G.and Harding,C.D.(1989)Int. ]. Syst. BacterioL 39,

217-223

43 Ray,S.K.,Johnson,M.C.and Ray,13.(1989)1. Ind MicrobioL 4, 163-171

44 Nielsen,I.W., Dickson,J.S.and Crouse,J.D. (1990)Appl. Environ. Microbiol. 56, 2142-2145 45 Daeschel,M.A.and Klaenhammer,T.R.119851AppLEnviron. MicrobioL 50, 1538-1541 46 Bhunia,A.K.,Johnson,M.C.and Ray,B. (1988)1. Appl. Bacteriol.65, 261-268 47 Gonzalez,C.F.and Kunka,B.5.(1987)Appl. Environ. Microbiol. 53, 2534-2538 48 Pucci,M.I.,Vedamuthu,E.R.,Kunka,B.S.and Vandenbergh,P.A. (1988)Appl. Environ. Microbiol. 54, 2349-2353 49 Okereke,A. and Montville,1.1.(I9911J. Food Protect. 54, 349-353 50 Reddy,S.G. Chen,M.L.and Patel,P.J.(1975)I. Food 5ci. 40, 314-318 51 Reddy,S.G., Henrickson,R.L.and OIson,H.C.(1970)I. FoodSci. 35, 787-791

52 Raccach,M. and Baker,R.C.(1978)I. Food Protect. 41,703-705

53 Raccach,M., Baker,R.C., Regenslein,I.M. and Mulnix, R.I. (19791 ]. Food Sci. 44, 43-46 54 Hanna,M.O., Savell,I.W., Smith,G.C., Purser,D.E., Gardner, F.A.and Vanderzant,C. (1983)J. Food Protect. 46, 216-221 55 Schillinger,U. and LOcke,F-K.(1986) Fleischwirtschaft66,

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Review Two recently developed ionization methods, electrospray ionization and matrix-assisted laser desorption, have improved the sensitivity and mass range of mass spectrometry. In particular, the techniques facilitate the analysis of fragile, high molecular weight molecules such as polypeptides and proteins. The new methods extend the applications of mass spectrometry in food research beyond those of the ion sources commonly used at present for large molecules (e.g. fast atom bombardment), and have been used to obtain mass spectra from bovine and human milk and other

New methods in the maSS spectrometry of proteins Ronald Beavls

biological fluids. obtained using FAB and PD to analyse molecules larger than l0 000 Da, but the studies have also demonstrated Mass spectrometry has had limited application as an the difficulties of examining such large, fragile analyacal technique for samples wP,h molecular masses molecules I-3. The simultaneous development of two new ion greater than 10 000 Da. The ion sources c o m m o n l y used for large molecules (fast atom bombardment, FAB; and sources, electrospray ionization (ESI) and matrixplasma desorption, PD) have been widely used for many assisted laser desorption (MALD) has led to a revoltypes of polar molecules, but the sensitivities of the ution in the mass spectrometry of large biopolymers. techniques rapidly decrease as the molecular mass of the These new techniques have improved the sensitivity of analyte increases. Some useful results have been analysis by three orders of magnitude, and have increased the mass range of mass spectrometry to include the analysis of molecules with masses greater than Ronald Beavis is at the Memorial Universityof Newfoundland,St John's, 2 0 0 0 0 0 D a . This review describes the technological bases of electrospray ionization and matrix-assisted Newfoundland,CanadaA1B 3×7.

Trends in Food Science & Technology October 1991

©1991,ElsevierSciencePublishersLid,(UK) Oq24-2244/91/$02.00

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laser desorption, and discusses practical aspects of using the methods to analyse polypeptides and proteins.

to interpret and the mass calculations can be performed automatically.

Electrosprayionization

Matrix-assistedlaserdesorption

The development of electrospray ionization has been covered in detail in several recent reviews 4-7. The possibility of using electrospray ionization to examine peptides and proteins was first demonstrated by John Fenn and his group at Yale University, USA and has subsequently been reproduced and expanded upon by Robert Smith's group at Pacific Northwest Laboratories, USA. The met.hod of producing ions is simple. The sample is dissolved in a polar solvent and a small amount of the solution is forced out of a narrow metal capillary tube held at high voltage (3-5 kV). The high electric field at the tip of the needle causes the solution to disintegrate into an aerosol plume of very small electrically charged droplets, a process commonly referred to as electrospray. The charged droplets are transported into a vacuum system and dried, using a series of differential pumped vacuum stages. The products of this drying procedure are neutral solvent molecules and the ionized analyte. The ions are then subjected to mass analysis, usually by a quadrupole mass spectrometer. The most important feature of electrospray ionization that allows the technique to be used for polypeptide research is that the ions formed may be multiply charged. When a multiply charged ion is introduced into a mass spectrometer, it produces a signal at an apparent mass that is an integer fraction of the actual mass of the ion, as a mass spectrometer measures an ion's mass-tocharge ratio (ralz), rather than the absolute mass of the ion. Multiple charging reduces the value of adz, allowing mass spectrometers with limited mtz detection ranges to be used to examine very large molecules. Most proteins will gain a sufficient number of charges such that an instrument that can measure mlz values in the range 1-2000 is usually sufficient to analyse molecules with absolute masses as large as 150 000 Da. Therefore, relatively inexpensive quadrupole mass spectrometers can be used. The high charges of polypeptide ions produced by electrospray ionization are the result of the properties of such molecules. Solutions used for electrospray are maintained at a low pH by adding a small amount of a weak acid (usually 1-5% acetic acid) to the solvent. in the acidic solvent, the polypeptide becomes a fully protonated, solution-phase ion with numerous positive charges (one charge per basic residue). Even after eleetrospray aerosol formation and subsequent drying, many of the protons are retained on the resulting gas-phase ions. Variability in the number of protons retained on a gas-phase ion leads to a distribution of ion charges and, therefore, a distribution of the observed mlz signals. A simple algorithm allows the actual charge (z) of a particular signal to be determined; the absolute mass (m) of the analyte molecule can then be calculated from the mlz and z values. While this process may seem a rather cumbersome way of measuring the mass of a large molecule, in practice, the signals obtained are quite easy

Pulsed lasers have been used to produce gas-phase ions from solid or liquid samples for over 20 years. The method suffered from the destructive effect that laser irradiation frequently had on the sample; numerous photolysis and pyrolysis products were observed during the ionization of samples of large biopolymers. Franz Hillenkamp and Micheal Karas at the University of Miinster, FRG discovered a method of sample preparation that minimized laser-induced degradation s. The analyte of interest (a polypeptide) was mixed with a large molar excess of a 'matrix', a substance chosen for its ability to absorb laser energy at a wavelength at which the analyte molecules are transparent. The gas-phase ions produced corresponded to the intact analyte molecules. Nicotinic acid was found to be a suitable matrix for proteins when using laser pulses with a wavelength of 266 nm. The gas-phase ions produced by matrix-assisted laser desorption are almost always either singly or doubly charged. In order to measure the large mlz ratios that result, a 'time-of-flight' mass spectrometer is usedg; ions of a particular charge are all given the same kinetic energy, and their masses are determined indirectly, from their velocities. Time-of-flight mass analysers have the advantages of unlimited mass range and very high ion transmission (most of the ions that enter the instrument strike the detector). A relatively complicated laser microprobe instrumenP ° was used by Karas and Hillenkamp for their initial experiments: subsequently, much simpler (and less expensive) instruments have been used, with improved results ~a'. The choice of matrix compound is a critical determinant of the quality of results. Nicotinic acid gave strong ion signals tbr pure proteins ~3, but ion signals were easily quenched by the presence of salts. Vanillic acid was found to be a good matrix for proteins, and was not as easily perturbed by contaminants~L Currently, the best results have been obtained using sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid, also referred to as sinapinic acid) ~4, which can be used with laser wavelengths ranging from 260 to 360nm (Ref. 15). Sinapic acid tolerates very high concentrations of salts and chaotropic agents in the sample; for example, it can selectively produce protein ions from a solution containing 5 mM protein and 1 M urea le.

Instrumentationand applications Both electrospray ionization and matrix-assisted laser desorption are currently the subject of intense research in a number of laboratories, and they should not be considered mature technologies. The potential of the techniques for protein analysis has led to the rapid development of commercial instruments that employ the new ionization methods. Most manufacturers of mass spectrometers currently offer an electrospray ion source as an option on tandem quadrupole mass spectrometers, Trends in Food Science& TechnologyOctober 1991

and a few offer the source alone. Only two manufacture~ currently offer laser desorption time-of-flight instruments. User-friendly devices that can be used by any scientist should be available within the next decade. Matrix-assisted laser desorption and electrospray ionization are used for the same purpose: to measure the molecular mass of polypeptides and proteins. Both techniques are very sensitive, requiring as little as ~1 picomole protein to produce a good signal. The techniques are, however, sufficiently different in detail that their applications to real-life problems will probably not overlap significantly. The mass resolution (or resolving power) of an instrument is a measure of its ability to distinguish between two adjacent signals; it is defined as the ratio of the mass (m) indicated by a signal peak to the width (Am) of the signal. Electrospray ionization provides relatively high mass resolution measurements of pure compounds (m/Am values may range from 500 to 1000), allowing the determination of molecular mass to within 0.01% (Ref. 7). The spectra produced by mixtures of polypeptides or proteins can be difficult to interpret, although software packages are currently available that attempt to automate the procedure of assigning the appropriate charge to a signal. In the case of mixtures, however, electrospray ionization can be easily coupled to reversedphase high performance liquid chromatography (HPLC), greatly increasing the applicability of the method to the analysis of mixtures of peptides. A tandem mass spectrometer with an electrospray ion source can be used to obtain sequence information about a peptide, although a complete sequence is rarely obtained. The inherent instability of the spray process can produce problems with some mobile phases, particularly those containing non-volatile buffers or detergents. Some types of posttranslational modifications, such as the addition of acidic sugars to a protein, can have an adverse effect on the intensity of the signals obtained. Matrix-assisted laser desorption using available timeof-flight mass spectrometers has a lower mass resolution (mlAm values in the range 100-400) than the instruments used with electrospray ionization, although the mass accuracy is similar (+0.01%) 16. However, it is possible to obtain high-quality mass spectra of proteins even in samples that have relatively high concentrations of impurities. Biological fluids, such as bovine or human milk ~7, apolipoprotein blood fractions ~7 and isolated food vacuoles from malaria parasites t8 have been analysed without purification. The method is insensitive to the primary sequence of a protein; positively charged proteins respond with signals that are similar to those of negatively charged proteins. High levels of glycosylation and other post-translational modifications do not alter the intensity of the signals obtained. A wide variety of solvents can be used in matrix-assisted laser desorption, including powerful protein solvents such as 7 0 ~ formic acid. These strengths and weaknesses have to be kept in mind when deciding which technique would be appropriate for a particular application. Hydrophobic proteins Trends in Food Science & Technology October 1991

and polypeptides that require strong solvents and detergents can only be analysed using matrix-assisted laser desorptio# 9, and proteins with a low abundance of charged amino acids (e.g. cereal grain prolamins) do not respond by electrospray, but produce strong signals after laser dcsorption. Mixtures of peptides obtained by trypsin digestion of a protein are naturally suited to electrosprzy ionization coupled with HPLC.

Future directions Research to increase the mass resolution of mass spectrometers used with matrix-assisted laser desorption ion sources is underway. Higher resolution time-or: flight instruments are being tested. Alternatively, it may be possible to couple laser desorption ion sources to instruments that have intrinsically high resolution and a wide m/z range, such as Fourier-transform ion cyclotron resonance mass spectrometers 2°. The use of new matrices may facilitate the analysis of other types of biopolymers, such as nucleic acids 2a and carbohydrates.

References 1 2 3 4 5 6 7 8 9

Barber,M. and Green, B.N. (1987) Rapid Commun. ,MassSpectrom. 1, 80-83 Chair,B.T. {1987) Int. L MassSpectrom. Ion Processes78, 237-250 Lacey,M.P. and Keough, T. (1989) Rapid Commun. MassSpectrom. 3, 323-328 Fenn,J.B., Mann, M., Meng, C.K., Wong, 5.F. and Whilehouse, C.M. (1990) Mass Spectrom. Rev. 9, 37-70 Fenn,J.B., Mann, M., Meng, C.K., Wong, S.F. and Whileho,se, C.M. (1989) Science 246, 64-71 Smith,R.D., Loo, I.A, Edmonds,C.G., Barina~a,CJ. and Udseth, H.R.

(1990)Anal Chem.62, 882-899 Mann,M. (1990) Org. MassSpectrom.25, 575-587

Karas,M. and Hillenkamp, F. (1988) Anal. Chem. 60, 2299-2301 Duckworth, H.E., Barber, R.C.and Venkatasubramanian,V.S. (1986) Mass Spectrometry(2nd edn), pp. 113-116, Cambridge University Press 10 White, F.A. and Wood, G.M. (1986) MassSpectrometry:Applications in Science and Engineering, pp. 82-88, John Wiley & Sons 11 Beavis,R.C. and Chait, B.T (1989) Rapid Commun. MassSpectrom. 3, 233-237 12 Salehpour,M., Perera, I. Kjellberg, J., Hedin, A., Islamian, M.A., Haakansson, P. and Sundqvist, B.U.R. (1989) Rapid Commun. Mass Spectrom. 3, 259-263 13 Karas,M., Bahr, U. and Hillenkamp, F. (1989) Int. I. Mass S~ctrom. Ion Processes92, 231-242 14 Beavis,R.C. and Chait, B.T. (1989) Rapid Commun. MassSpectrnm. 3, 432-435 1S Beavis,R.C. and Chair, B.T. (1989) Rapid Commun. Mass Spectrom.3, 436-439 16 Beavis,R.C. and Chait, B.T. (1990) Anal. Chem. 62, 1836-1840 17 Beavis,R.C. and Chait, B.T. (! 990) Proc. Nail Acad. 5ci. USA 87,

6873-6877 18 Goldberg,D.E, Slater,A.F.G.,Beavis,R.C.,Chait, B.T.,Cerami,A. and Henderson,G.B.(1991)]. Exp.Meal.173, 961-969 19

20

Perera,I.K., Candy, I.M., Haakansson,P., Oakley, A.E., Brinkmalm, G. and Sundqvist, B.U.R. (1990) Rapid Commun. Mass 5peOrom. 4, 527-532 Hettich, R.L. and Buchanan,M.V. (1990) I. Am. Soc. Mass Spectrom.2,

22-28 21 Nelson,R.W.,Thomas,R.M.and Williams,P. (1990) RapidCommun. Mass Spectrom.4, 348-355

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