432

PEPTIDES AND PROTEINS

[23]

[23] S a m p l e P r e p a r a t i o n for P l a s m a D e s o r p t i o n Mass Spectrometry B y PETER ROEPSTORFF

Sample amounts are often very limited in practical biochemical studies, and the sample quantity available for mass spectrometric analysis may represent tedious and costly preparation procedures, or may even be the ultimate sample amount. It is, therefore, of the utmost importance that the mass spectrometric procedures employed give optimal chances for success at the first trial, and extract maximum information from a given sample amount. Plasma desorption mass spectrometry (PD-MS) j'2 has recently been very successful for the analysis of peptides and proteins up to a molecular weight of approximately 35,000, 3 and it seems to be a promising routine method in the protein chemistry laboratory, because the instrumentation is relatively inexpensive and very simple to operate. 4 Because of this simplicity the only way to improve the results is by improving the quality of the sample. The method by which the sample is prepared is, therefore, of major importance. As in all desorption ionization methods, the presence of low-molecular weight contaminants, and among these especially alkali metal salts, may reduce the spectrum quality considerably, and often leads to complete suppression of the signals from high-molecular weight samples. Unfortunately, most methods in protein chemistry are based on the use of salt-containing aqueous buffers, necessitating the development of sample application methods and alternative solvent systems, acceptable for use in mass spectrometry. The PD mass spectra of peptides and proteins most frequently contain only molecular ions and no structurally meaningful fragment ions. It is, however, possible to obtain structural information by combining chemical or enzymatic cleavage or modification procedures known from classical protein chemistry with the mass spectrometric analysis. This chapter will describe the methods currently used in protein studies I D. F. Thorgerson, R. P. Skowronski, and R. D. Macfarlane, Biochem. Biophys. Res. Commun. 60, 616 (1974). 2 R. D. Macfarlane, this volume [11]. 3 For a recent review see R. J. Cotter, Anal. Chem. 60, 781A (1988). 4 Commercial instruments are available from Biolon Nordic AB, Box 15045, S-750 15 Uppsala, Sweden.

METHODS IN ENZYMOLOGY, VOL. 193

Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.

[23]

SAMPLE PREPARATION FOR PLASMA DESORPTION M S

433

3-7 kV

\ Capil.tary 5-20.ut

.,:,,, ..... J ' i

i

, i

,"

i

Focusing =~'-- Voltage Ii

fl

,,~-----AI

foit

'2 FIG. 1. Principle of the electrospray method for sample application.

by PD-MS in the author's laboratory for sample application and for obtaining structural information on the sample.

Sample Application by Electrospray The first method used for sample application in PD-MS was the electrospray method, 5 the principle of which is shown in Fig. 1. Procedure

Sample solution (3-10/zl) containing 0. I-10/~g//xl of peptide is placed in the capillary and the high voltage gradually increased until the spray begins (visual observation in a parallel light beam). The focusing voltage is then regulated to obtain a well-defined sample deposition on the aluminum foil with a spot-size of the area exposed to fission fragments (approximately one-half the diameter of the aluminum foil). The spray stops when all of the sample solution is consumed. 5 C. J. McNeal, R. D. Macfarlane, and E. L. Thurston, Anal. Chem. 51, 2036 (1979).

434

PEPTIDES AND PROTEINS

[23]

Comments

Electrospray is only possible with reasonably volatile organic solvents, e.g., acetic or trifluoroacetic acid, chloroform, acetone, methanol, or ethanol. A water content up to 30% may be acceptable. This limits its applicability in protein chemistry because most proteins are best dissolved in aqueous solvents. The electrospray technique, furthermore, requires samples of a very high purity with respect to low-molecular weight impurities and considerably higher sample amounts than the nitrocellulose-based techniques described below. Improved results can be obtained by cospraying peptide and protein samples with an excess of reduced glutathione (GSH). 6 This results in improved molecular ion yields, increased abundance of multiply charged molecular ion species, and often also in a reduction of the influence of alkali metal ions. GSH must be present in a 25-100 times molar excess relative to the sample. In practice, this is achieved by making the spray solvent 50 mM with respect to GSH. Typically, the sample is dissolved in a solution of 50 mM GSH in acetic acid/trifluoroacetic acid, 10 : 1 (v/v), to a concentration of 1 /xg//zl; 3-7/xl is electrosprayed on the aluminum target. Sample Application by Adsorption on Nitrocellulose A considerable improvement in sample handling procedures has been obtained by the introduction of nitrocellulose as a matrix. 7 The principle (see Fig. 2) is based on adsorption of the protein or peptide to a thin nitrocellulose layer placed on the aluminum foil, followed by removal of salt contaminants by washing with ultrapure water or dilute acid. The use of nitrocellulose compared to electrospray results in improved molecular ion yields (Fig. 3), increased abundance of multiply charged molecular ions, and sharper peaks. The effect is qualitatively similar, but quantitatively better, than that obtained by adding GSH to the electrospray solution. Three different methods have been used for application of the sample onto fhe nitrocellulose layer. The original method was based on adsorption of the protein from a few microliters of aqueous solution followed by washing of the surface with 1-2 ml of pure water or dilute acid (Fig. 2, left-hand side). The sample solution is sandwiched between the nitrocellu6 M. Alai, P. Demirev, C. Fenselau, and R. J. Cotter, Anal. Chem. 58, 1903 (1986). 7 G. P. Jonsson, A. B. Hedin, P. L. Hhkansson, B. U. R. Sundqvist, G. S. S/ive, P. F. Nielsen, P. Roepstorff, K. E. Johansson, I. Kamensky, and M. S. L. Lindberg, Anal. Chem. 58, 1084 (1986).

[23]

SAMPLE PREPARATION FOR PLASMA DESORPTION MS

TARGET

1-s~t *-"-*I

Drop- deposition

Cover

Distribution

. , . Nitrocellulose . . . . . . . . . . . i1 "='Aluminized polyester ..............~b-Brass ring

Dipping

glass

435

~.~ © Volume ~ O.Smt

Spin-drying

I-s~t//

Sampl e on appl i c ati step

T Spinning target

~/ I - 2 x O.5-1mt Extensive rinse

~] 1-2xlmt Dip- rinse

~ /

Ristep nsing

I - 2 x 5-10Opt Micro-rinse

FIG. 2. Principle of the nitrocellulose adsorption method and methods for preparing the sample. Left, adsorption followed by extensive wash; center, the dipping technique; right, the spin technique.

lose layer and a microscope cover glass in order to spread it on the surface. This application technique requires sample concentrations of the order of 0.5 to 1/zg//zl. An alternative method, which allows application from very dilute solutions, is to dip the nitrocellulose-covered target in the protein solution for 0.5 to 1 min, followed by drying and washing of the surface (Fig. 2, center). This method has, for example, been successfully used to extract lutenizing hormone releasing hormone from a 5 × 10 - 7 M solution. 7 The third method, now the standard in the author's laboratory, applies the sample on a spinning nitrocellulose target 8 (Fig. 2, right-hand side). This method was first introduced for application of small peptides, which do not bind strongly to nitrocellulose, and therefore might be removed in the washing procedure. The spin-drying procedure combines the adsorption and washing in a single step, because a thin sample layer is deposited in the central area (the area exposed to fission fragments), whereas the excess 8 p. F. Nielsen, K. Klarskov, P. HCjrup, and P. Roepstorff, Biomed. Environ. Mass Spectrom. 17, 355 (1988).

436

PEPTIDES AND PROTEINS

[23]

d

MH + yield 1.6 ~ "

"

"

NC(ew)

1.4

1.2

1.0

0.8

0.6

0.4

a~a

1 0.2

.J

//':-°

a

[5

I> 100

101

102

103

10'

10s pmol insutin

FIG. 3. Molecular ion yield of insulin (expressed as number of molecular ions recorded per 100 fission events) as a function of the sample amount and sample application technique. &, Spin drying (sd) on nitrocellulose (NC); A, adsorption on nitrocellulose followed by extensive wash (ew); I7, electrospray with glutathione (G); and O, electrospray (E). (Adapted from Ref. 12 with permission from the publisher.)

sample and the more soluble salts migrate with the solvent to the periphery of the target. The spin-drying method has been found to give better sensitivity, not only for small peptides and for other types of compounds, which do not bind to nitrocellulose, but also for large peptides and proteins (Fig. 3). The following sections describe the procedures currently used as standard in the author's laboratory.

Preparation of Nitrocellulose Targets A stock solution of nitrocellulose in acetone (2/zg//zl) is prepared by dissolving a small piece of nitrocellulose blotting membrane (Bio-Rad Laboratories, Richmond, CA) in an appropriate volume of acetone (analyt-

[23]

SAMPLE PREPARATION FOR PLASMA DESORPTION MS

437

ical-grade). This solution (25-50/~1) is electrosprayed onto the aluminized Mylar target. The focusing voltage is adjusted to give a spot size of approximately 7 mm in diameter. Each target is visually inspected for homogeneity. A thick homogeneous nitrocellulose layer is increasingly important with increasing molecular weight of the sample, or for the analysis of very small sample amounts. The best targets are, therefore, used for such samples, whereas lower quality targets may be used for less critical samples.

Application of Sample onto Nitrocellulose The peptide or protein may, in principle, be dissolved in any solvent which does not cause damage to the nitrocellulose film, e.g., water, dilute acid or base, salt-containing buffer solutions, 2-propanol and ethanolwater or acetonitrile-water mixtures. The most frequently used solvent in the author's laboratory is ultrahigh-quality (UHQ) water (see Comments) containing 0.1% trifluoroacetic acid (TFA) and 15% ethanol or acetonitrile. If necessary, the pH may be adjusted by addition of ammonia. The nitrocellulose-covered target is placed in a holder mounted horizontally on the shaft of a variable-speed motor. The sample solution (2-5/~1) containing 0.01-1/~g//,d of sample is placed in the center of the target, and the motor speed is gradually increased to distribute the solvent on the entire surface, followed by drying at full speed (2500 rpm). The drying, which propagates from the center, is easily observed. Sometimes the sample solution is simply allowed to dry without spinning, and distribution on the surface omitted or effected in the following washing step. From Fig. 3 it is seen that the application of washing procedures may lead to reduced sensitivity, probably due to removal of sample in the washing step. On the other hand, application of too much sample by the spin technique leads to reduced molecular ion yields. Fortunately, PD-MS is essentially a nondestructive method, which means that it is possible to record a spectrum of the sample after application, for example, by the spin technique. If indicated upon examination of the spectrum, a washing procedure may then be applied and the sample reanalyzed. A poor sample ion yield indicates the need for washing. Most frequently, poor yields are due to a too high content of alkali metal ions, which can be ascertained by observation of the ions for Na + and K + at m/z 23 and 40, respectively. If the summed abundance of these ions is more than one-half of the abundance of the H ÷ ion, washing is indicated. Another reason for low sample ion yield may be that too much or too little sample has been applied. If the former is suspected, washing may also improve the result, whereas improved results in the latter case can only be obtained by prolonged recording of the spectrum in order to obtain better ion statistics.

438

PEPTIDES AND PROTEINS

[23]

Washing

Washing of the nitrocellulose-bound sample may be performed by either of the methods shown in Fig. 2. Microwashing, performed by applying 2 x 5-10 /xl of a 0.1% TFA solution to a slowly spinning target, followed by drying at full speed, is usually preferred. If indicated by a high content of alkali metal ions, or if a salt-containing sample buffer has been used, washing with 2 x 100/xl of washing solution on a spinning target may be preferable, or an extensive wash may be applied. The latter is effected by literally sluicing the nitrocellulose surface with 2 x 0.5 to 1 ml of solvent with a Pasteur pipette as illustrated in Fig. 2. Comments

The quality of the water used for the sample solution, the washing solvents, and also for the HPLC solvents, if HPLC is the last purification step prior to mass spectrometric analysis, is of the utmost importance. In the author's laboratory, 15-18 M12/cm resistivity water (UHQ-water) prepared with an Elgastat UHQ apparatus (Elga Ltd., High Wycombe Bucks, UK) is always used.

In Situ Reactions to Obtain Further Information

Plasma desorption mass spectra of peptides and proteins are dominated by molecular ion species, whereas structurally meaningful fragment ions are sparse or absent. However, as mentioned above, most of the sample is undamaged after recording a spectrum. The remaining nitrocellulosebound sample can, therefore, after removal of the target from the mass spectrometer, be subjected to chemical or enzymatic reactions in situ, 9"1° and structural information thus be obtained. The most commonly used reactions are reduction of disulfide bonds with dithiothreitol (DTT), 9'11'12 cleavage with endopeptidases, ~°'12'13 resulting in so-called PD maps, and cleavage with exopeptidases, especially carboxypeptidases, 1°'14which can 9 B. T. Chait and F. H. Field, Biochem. Biophys. Res. Commun. 134, 420 (1986). 10 B. T. Chait, T. Chaudhary, and F. H. Field, in "Methods in Protein Sequence Analysis 1986" (K. A. Walsh, ed.), p. 483. Humana, Clifton, NJ, 1987. 11 p. F. Nielsen and P. Roepstorff, Biomed. Enoiron. Mass Spectrom. 18, 138 (1989). 12 p. F. Nielsen, P. Roepstorff, I. G. Clausen, A. B. Jensen, I. Jonassen, A. Svendsen, P. Balschmidt, and F. B. Hansen, Protein Eng. 2, 449 (1989). 13 p. Roepstorff, P. F. Nielsen, K. Klarskov, and P. HCjrup, in "The Analysis of Peptides and Proteins by Mass Spectrometry" (C. J. McNeal, ed.), p. 55. Wiley, New York, 1988. 14 K. Klarskov, K. Breddam, and P. Roepstorff, Anal. Biochem. 1811, 28 (1989).

[23]

SAMPLE PREPARATION FOR PLASMA DESORPTION M S

439

give C-terminal sequence information. An example of an in situ reduction is shown in Fig. 4. Procedures

Reduction of disulfide bonds is effected by placing 2/zl of a solution of 0.08 M DTT in 0.1 M ammonium bicarbonate, pH 7.8, on the sample surface. A microscope cover glass is placed on top to prevent evaporation, or the target is placed in a small, closed plastic box containing a moist piece of filter paper. After reaction at room temperature for 10 min, the target is spin-dried and reinserted into the mass spectrometer. Enzymatic cleavages are performed in a similar way. In order to obtain acceptable cleavage yields in a reasonably short time, it is necessary to use a high enzyme-to-substrate ratio, e.g., between 1 : 2 and 1 : 1. Typical standard reaction conditions are with trypsin, Staphylococcus aureus protease, or carboxypeptidases: 15-30 min at 37° with 2 /xl of a solution containing 1/xg//zl of enzyme in 0.1 M ammonium bicarbonate, adjusted to an appropriate pH with acetic acid.

Monitoring of Reactions in Solution In situ reactions are often incomplete, most likely because some sample molecules embedded in the matrix are inaccessible to the reagents, or because the enzymes are inactivated by contact with the nitrocellulose. Therefore, if sufficient sample amounts are present, it may be advantageous to monitor reactions carried out in solution. Likewise, mass spectrometric monitoring of a preparative reduction and alkylation reaction or an enzymatic cleavage might often be useful. ~3'~5 Procedures

The standard procedures used in protein chemistry can normally be applied. It is, however, preferable to exchange alkali metal salts in buffers with ammonium salts, because the latter do not have a negative effect on the quality of the plasma desorption spectra. 16If this is possible, aliquots of 2 to 10/~1 are withdrawn from the reaction solution and applied onto a nitrocellulose target by the spin-drying procedure, followed by mass t5 p. Roepstorff, P. F. Nielsen, K. Klarskov, and P. HCjrup, Biomed. Environ. Mass Spectrom. 16, 9 (1988). 16 M. Mann, H. Rahbek-Nielsen, and R. Roepstorff, in IFOS V, "Proceedings of the Fifth Symposium on Ion Formation from Organic Solids" (A. Hedin, B. U. R. Sundquist, and A. Benningshoven, eds.), p. 47. Wiley, New York, 1990.

440

PEPTIDES AND PROTEINS

[23]

1000

A ÷÷

MH 2

i 50¢

" " ' O . . . . . . . . . . . . . . . . . . . . . . .

1225

1.

. . . . . . . . . . .

.

IL~kLJ, i l u J .....

2874

LI

,'.."

4523

u Jk,I rr[ .... ..~' .....

.•.,•.,JL.=.= i- . . . . . . . .

bin=2ch lch=lns

6173

M/Z

2000

B MH~B-CHAIN)

100(

++

MH2(B-CHAIN) MH~A-CHAIN)

bin=2ch i

.

.

.

.

.

.

.

.

.

i

.

.

1223

.

.

.

.

2865

.

.

.

.

.

.

r ' '

4515

6161

M/Z FIG. 4. Positive ion PD-mass spectra of human insulin showing the intact molecule (A) and the same sample after in situ reduction showing the A and B chains (B).

[24]

MOLECULAR WEIGHT ANALYSIS OF PROTEINS

441

spectrometric analysis. UHQ-water is, of course, used for all solvents. If metal salts cannot be eliminated, or if the solution contains high concentrations of urea or guanidine-HCl, extensive washing is required, a procedure that may lead to loss of small peptides.

[24] M o l e c u l a r W e i g h t A n a l y s i s o f P r o t e i n s By IAN JARDINE

Introduction The molecular weight of a protein has always been recognized as an important analytical parameter in biochemistry. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is universally used to purify proteins; molecular weights are routinely determined after separations by comparison of the migration of the protein of interest to that of a set of standard proteins. A typical protein molecular weight experiment would be to analyze the molecular weight before and after removal of carbohydrate to estimate the carbohydrate content of the glycoprotein and the mass of the deglycosylated protein. Estimates of the accuracy of SDS-PAGE protein molecular weight determination range from a few percent for well-behaved proteins in this system up to 20 or 30% for heavily glycosylated proteins. Clearly, high accuracy (e.g., to < 1.0%) in protein molecular weight measurement has never been achieved with SDS-PAGE, so even some of the molecular weight standards used are not well characterized. After many years of effort to ionize, vaporize, and, subsequently, mass analyze proteins at high sensitivity via ionization techniques such as plasma desorption (PD) and fast atom bombardment (FAB), the protein molecular weight determination scenario has now changed dramatically with the recent discovery of two new highly sensitive mass spectrometric ionization techniques, electrospray (ESI) 2,3 and matrix-assisted laser desorption (LD). 4,5 These methods have both been demonstrated to allow i H. D. Kratzin, J. Wilffang, M. Karas, V. Neuhoff, and N. Hilschmann, Anal. Biochem. 183, 1 (1989). 2 C. M. Whitehouse, R. M. Dryer, M. Yamashita, and J. B. Fenn, Anal. Chem. 57, 675 (1985).

3C. G. Edmonds and R. D. Smith, this volume [22]. 4 M. Karas, U. Bahr, A. Ingendoh, and F. Hillenkamp, Angew. Chem., Int. Ed. Engl. 28, 760 (1989). 5 F. Hillenkamp and M. Karas, this volume [12]. METHODS IN ENZYMOLOGY,VOL. 193

Copyright © 1990by Academic Press, Inc. All rightsof reproductionin any form reserved.