Anal. Chem. fS90, 62,1532-1536
1532
Table I. Three-way Anova of log-Transformed Data from Reference 2
source of variation mean samples catalysts laboratories samples X catalysts samples x laboratories catalysts x laboratories samples X catalysts x laboratories error total
sum of squares 27879.3 1054.5 0.00196 0.4747 0.0120
degrees of freedom 1
25 1 21 25
F mean square ratio 27879.3 42.2 0.00196 1.34 0.0226 43.6 0.000480 1.65
0.2996
525
0.000571
1.10
0.0307
21
0.00146
2.82
0.1526
525
0.000291
0.56
0.5931 28935.4
1144 2288
0.000518
protein determinations. The three-way anova of the logtransformed data provides no justification for doing this. The hypothesis that the adjusted treatment means for laboratories, Y..k.- Y...., constitute a sample from a normal population is easily accepted by the Shapiro-Wilk test (6). Removing laboratory treatments with the largest biases will result in serious underestimation of the variance of the laboratory effects and, hence, of the “reproducibility variance” of the analytical method (7). Thompson found that robust estimates of sample variances were noticeably greater than those computed by Kane and suggested that Kane’s outlier tests gave a significant proportion of type I errors. A more serious problem is that both Thompson and Kane sought to correct for discordance without making allowance for laboratory bias. When the linear model was collapsed to include only the main significant effects Yik= M + Si Lk eik and the AnscombeTukey test (8)for discordant residuals was applied with a 2.5% risk premium, 43 outliers were identified in the first cycle of iteration and one in the second. This compares with 117 outliers found by Kane visually from the scatter of points in a Youden plot (7). While so small a proportion of outliers should not overtax the robustness of the anova against errors in hypotheses testing, there can be a profound effect on the components of variance in terms of which the variability of an analytical method is expressed. Although the 44 outliers represent only 2% of the data, by their removal the error variance in Table I, estimated by the method of Healy and Westmacott (9), is approximately halved.
+ +
The power of the anova will be less if the data are inhomogeneous of variance, and Kane’s data are decidedly so: there is a clear dependence of the mean absolute difference between replicates on the protein level. Square-root and logarithmic transformations were tried to achieve homogeneity of variance, and the latter was found satisfactory by the Levine-median test, a robust and powerful test for homoscedasticity and itself an anova procedure ( 4 ) . Formulas for computing and comparing mean squares, with In (Y,jkJ as the random variable, were taken from Ostle and Mensing (5). The results are shown in Table I. The anova results prove that there is no overall catalyst effect; however, the F ratio for the catalyst-laboratory interactions would occur with a probability of less than 0.001, so it is likely that there are real differences between the two catalysts in particular laboratories. Also, the F ratio for the catalyst-sample interactions is marginally significant ( P = 0.025), so there is reason to doubt that both catalysts work equally well with all types of feeds studied. This is consistent with Kane’s finding of a significant method effect, at the 95% confidence level, with four of the feed samples. However, Thompson’s analysis of the data finds no matrix effects for particular materials. There is a marked laboratory effect owing to the critical nature of the Kjeldahl digestion parameters as noted by Kane who, prior to his own data analysis, discarded the results of three laboratories which were consistently high or low in their
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9)
Thompson, Michael. Anal. Chem. 1989, 61, 1942-1945. Kane, Peter F. J. Assoc. Off. Anal. Chem. 1984, 6 7 , 869-877. Box, George E. P. Biomehka 1953, 40, 318-335. Brown, M. B.; Forsythe, A. B. J. Am. Statist. Assoc. 1974, 69, 364-367. Ostle, B.; Mensing, R. W. Statistics in Research; The Iowa State University Press: Ames, IA, 1975; Chapter 10. Shapiro, S.S.; Wilk. M. B. Biomehlka 1965, 52, 591-611. Youden, W. J.; Steiner, E. H. Statistical Manual of the AOAC; AOAC: Arlington, VA, 1975. Anscombe, F. J.; Tukey, J. W. Technometrics 1983, 5 , 141-160. Healy, M. J. R.; Westmacott, M. H. Appl. Stat. 1956, 5 , 203-206.
Neil E.Jones Michigan Department of Agriculture Laboratory Division East Lansing, Michigan 48823
RECEIVED for review November 27, 1989. Accepted March 29, 1990.
Background Emission from the Peroxyoxalate Chemiluminescence Reaction in the Absence of Fluorophors Sir: Peroxyoxalate chemiluminescence has been effectively used as a very sensitive postcolumn detector for HPLC in analyses of fluorescent analytes or fluorescent-labeled analytes including dansylated amino acids ( I ) , fluorescamine-labeled catecholamines ( 2 ) , 7-fluorobenzo-2-oxa-1,3-diazole-4sulfonate-labeled thiols (3), 4-fluoro-7-nitrobenzo-2-oxa-1,3diazole-labeled primary and secondary amines ( 4 ) ,PAHs (5), amino-substituted PAHs ( 6 ) ,and 3-aminofluoranthrene derivatives of aldehydes and ketones (7). The fluorescent species is chemically excited by a transfer of energy from an intermediate formed by the oxidation of
an oxalate derivative. Since not all fluorophors are efficient chemilumiphors, a selectivity advantage is possible in analytical applications. An additional analytical advantage is improved detection by elimination of source related problems including stray light and fluctuations in intensity. However, detection by chemical excitation is ultimately limited by background chemiluminescence observed without fluorophor present. When peroxyoxalate chemiluminescence detection is used as a postcolumn HPLC detector, the background emission is observed as a voltage offset above the photomultiplier tube
0003-2700/00/0362-1532$02,50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62,NO. 14,JULY 15, 1990
(PMT) dark current. With bis(2,4,6-trichlorophenyl)oxalate (TCPO) as the oxalate ester, the background emission generates about as much current in a P M T as the dark current a t a given PMT voltage setting (8). Slight pulsations in the flow system lead to small variations in the reagent concentrations, which result in a changing amount of background emission observed as base-line noise. Sigvardson and Birks have shown that the spectrum of the background emission from TCPO and hydrogen peroxide is independent of solvent, making fluorescent impurities an unlikely cause (5). This suggests that an independent chemiluminescent pathway may be responsible for the low-intensity background emission. The background emission may be linked to the formation and decomposition of reaction intermediates. Several reaction schemes have been proposed for the fluorophor-induced chemiluminescence. Each proposed chemiluminescent mechanism differs in either the identity or the number of key intermediates responsible for the excitation of the fluorophor. Rauhut proposed several intermediates but suggested that 1,2-dioxetanedione is the key intermediate (9). Palmer proposed a dioxetanone as the key intermediate (10). On the basis of kinetic studies, Givens has proposed that the peroxyoxalate reaction may produce an excited fluorophor by two parallel mechanistic pathways with more than a single key intermediate (11). Background chemiluminescence from reactive intermediates may be consistent with any of these reaction mechanisms. The background chemiluminescence produced by using several different peroxyoxalate reagents and hydrogen peroxide has been investigated. Possible chemiluminescent intermediates that may be responsible for the background emission are considered on the basis of on spectral evidence. Also, the limitations of the method in trace determinations of fluorescent analytes is examined.
EXPERIMENTAL SECTION Chemicals. Bis(2,4,6 trichlorophenyl) oxalate (TCPO) and bis(2,4-dinitrophenyl) oxalate (DNPO) were synthesized and purified following the procedure of Mohan and Turro (12). Bis[l-(1H)-2-pyridoxyl]glyoxal(BPG) was prepared according to the reported procedure (13). Bis(2,4,5 trichloro-6-carbopentoxyphenyl) oxalate (CPPO) was donated by A. Mohan (New Jersey Department of Health). Hydrogen peroxide (90%) was obtained from MCB. HPLC grade ethyl acetate was used as solvent (Baker). The fluorophor 16,17-diheptanoyloxyviolanthrone was obtained from Aldrich. Emission Spectra. Emission spectra were recorded by using a Fluorolog 2+2 spectrofluorimeter with the lamp source off. Emission slits were set at 8 mm (14-nm band-pass). A spectrum was obtained for a single scan by photon counting for 0.5 s at intervals of 10 nm. Ten scans were averaged for each spectrum reported. Except for the spectra of 16,17-diheptanoyloxyviolanthrone (DHV), all spectra have been corrected for wavelength-dependent response of the photomultiplier,the bandwidth of the monochromater, and the transmission of the monochromater. Saturated solutions of TCPO (0.045 g/mL), DNPO (0.020 g/mL), and BPG (0.020 g/mL) were added to a 1-cm cell and mixed with 100 p L of 70% hydrogen peroxide. The spectra were recorded. The spectra of hydrogen peroxide and CPPO (0.17 g/mL) and of a saturated solution of CPPO (0.39 g/mL) were recorded as above. The fluorophor was diluted in ethyl acetate, and the spectrum was recorded by the addition of DNPO and hydrogen peroxide as above. Quantitative ChemiluminescenceMeasurements. A TD20E luminometer (Turner Designs) with strip chart readout on a Fisher Series 5000 recorder was used. Polypropylene test tubes (1.6 mL 8 X 50 mm) were used to test all samples. A 100-pL portion of 8020 acetonitri1e:l mM phosphate buffer (pH 8.5) was mixed with 100 pL of 1 M hydrogen peroxide in
1533
5 .
300
700 WAVELfNGTHInml
spectra of TCPO (-), DNPO (- - -), CPPO (. and at 0.01-0.05 g/mL reagent and hydrogen peroxide. The intensity range in relative units is (0-2.00) X lo4 for TCPO, (0-2.50) X lo4 for DNPO, (0-8.00) X lo3 for CPPO, and (0-6.70)X lo2 for BPG. Figure 1. Emission
BPG
e),
(-e-)
acetonitrile and either 10 pL of 9.2 X 10” M DHV in acetonitrile (sample) or 10 pL of acetonitrile (blank). A 100-pLinjection of 4 mM TCPO in ethyl acetate was made, and the emission followed for 2 min. Initial reagent concentrations were 1.3 mM TCPO, 0.32 M hydrogen peroxide, and 3.0 X lo-’ M DHV. Almost no light was emitted after 2 min. Sample and blank were run with and without a 500-nm filter inserted in front of the PMT. Quenching was investigated by adding 10 pL of methylene chloride to the above system before the TCPO was injected. Nitrogen purging of the hydrogen peroxide solution for 5 min before use was also tried.
RESULTS AND DISCUSSION Spectra of Peroxyoxalate Background Chemiluminescence. A broad featureless emission band with a maximum around 450 nm is observed in the chemiluminescence spectra of 1-470 solutions of DNPO,TCPO, CPPO, and BPG (Figure 1). A band-pass of 10 nm was used to increase throughput, but fine features may not be resolved in the spectrum. The spectrum from the reaction of TCPO and hydrogen peroxide is similar to the emission spectrum of TCPO and hydrogen peroxide previously reported (5). A second low-intensity peak a t 540 nm is observed when TCPO and hydrogen peroxide are mixed. In addition to the major emission at 450 nm, the spectrum of background emission produced by DNPO contains a broad shoulder between 575 and 700nm. No second peak is observed in the BPG spectrum, but this background spectrum has the lowest signal-to-noise ratio of the reagents studied, so a small secondary peak may not be observable. A low-intensity peak in the emission of CPPO and hydrogen peroxide is observed a t 620 nm (Figure 2). When the concentration of the CPPO is increased to about 0.4 g/mL, the intensity of the longer wavelength emission increases and then decreases at a rate faster than the emission at 450 nm. Under experimental conditions, the long-wavelength emission reaches maximum intensity in about 3-4 min and then decreases in intensity, while the 450-nm emission continues to increase in intensity during the experiment. The behavior of the emission in saturated CPPO solutions suggests the existence of two emitting species with different rates of formation and decomposition. The species emitting light at 620 nm is more quickly formed and decomposed, while the species responsible for the 450-nm emission is formed relatively slowly. In Givens’ experiments, the two time-dependent maxima are observed only when an approximately equal molar concentration of ester and hydrogen peroxide is present initially. A large excess of either
1534
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990 Scheme I TCPO
WAVILINGTH’nm,
Figure 2. Emission spectra of CPPO (0.39 g/mL) at various times after mixing with hydrogen peroxide.
reagent leads to single f i t - o r d e r decay kinetics. In this study, two maxima are observed for CPPO and hydrogen peroxide when in approximately equal molar concentrations. In the determination of the background spectra of saturated CPPO solution, approximately equal molar quantities of CPPO and hydrogen peroxide were used. The experiment could not be repeated with TCPO, DNPO, and BPG at high concentration because of the limited solubility of these reagents in ethyl acetate. Decreasing the amount of hydrogen peroxide, to achieve an equal molar ratio of ester and hydrogen peroxide, results in unobservable intensity of light emission. When CPPO concentrations of 0.03-0.10 g/mL are used, the emission at 620 nm is still observed, but the low intensity of the 620-nm emission a t these CPPO concentrations makes study of the time-dependent behavior difficult. T o determine if any reagents or reaction products were fluorescent, all components were evaluated for fluorescence. Each solution of DNPO, TCPO, CPPO, and BPG was examined for fluorescent components by scanning the excitation monochrometer from 240 to 430 nm while monitoring the emission at 450 nm with 10-mm slit openings. No fluorescence emission was observed from the peroxyoxalate reagents and impurities. The reaction products were also examined for fluorescence emission. The phenols 2,4,6-trichlorophenol and 2,4-dinitrophenol are produced from the decomposition of TCPO and DNPO, respectively. If the reaction products are responsible for the emission, addition of these products to the reaction mixture would change the spectrum. When these phenols were added to the respective oxalate ester and hydrogen peroxide, the emission was similar to that observed without the addition of phenol. These results indicate that the background light emission is not caused by fluorescence from the peroxyoxalate reagent impurities or phenolic products of the reaction. The fluorescent species must be formed during the reaction. Interpretation of the Spectra with Respect to Chemiluminescent Mechanisms. Previous investigations of the peroxyoxalate chemiluminescent mechanism are based on data generated with fluorophor present, but relatively little information has been reported on the emission in the absence of fluorophor. Analysis of the background spectra with respect to the proposed peroxyoxalate mechanisms may lead to additional information about the nature of the mechanism or the intermediates involved. Furthermore, the analytical limitations of the reaction may be assessed from analysis of the background spectra. The 1,2-dioxetanedione species, which has been proposed as an intermediate in peroxyoxalate chemiluminescence, eventually decomposes to carbon dioxide. Excited singlet and triplet 1,2-dioxetanedione have been proposed as the source
+
H202 E5N +
of the background emission (14). However, the differences in the background spectra of the peroxyoxalate esters with hydrogen peroxide cannot be explained by a single common intermediate. Recently, a number of intermediates have been proposed other than dioxetanedione. Palmer (10) suggests a dioxetanone intermediate that contains the phenolic portion of the original peroxyoxalate reagent. If such an intermediate were responsible for the background emission, a different spectrum for each peroxyoxalate reagent might be observed. The time-dependent nature of the background spectrum of CPPO is inconsistent with a proposed single intermediate. It is not known whether the spectra of TCPO, DNPO, and BPG with hydrogen peroxide would also show time dependence with appropriate reagent concentrations. In systems where hydrogen peroxide is in large excess, a single major emission would be consistent with a single major intermediate under these conditions. Steinfatt (15) has reported that excited atomic oxygen is the active intermediate mainly responsible for energy transfer in peroxyoxalate systems. The excited oxygen is generated from an unstable oxalic acid peroxyanhydride. Since excited atomic oxygen does not have an emission around 450 nm, the background emission is not due to this species. Excited singlet molecular oxygen does have emissions at 633 and 703 nm, but these are not observed in the peroxyoxalate system (16). On the basis of kinetic studies, Givens has proposed that the peroxyoxalate reaction may produce excited fluorophor by two parallel mechanistic pathways (Scheme I) (11). The identity of the intermediates x , x ’, y , y’, and z are not known. Background emission from two intermediates in a complex kinetic scheme given above can explain the time-dependent nature of the spectrum from CPPO and hydrogen peroxide: each intermediate may be formed and decomposed at different rates. It should be noted that this observation was made with equal molar concentrations of ester and hydrogen peroxide similar to the conditions in Givens’ work. These conditions could not be duplicated with TCPO, DNPO,and BPG because of the solubility limitations as previously stated. Furthermore, a common intermediate in all four reagents may be responsible for the major emission at 450 nm. Because the emission at 450 nm is similar for all four peroxyoxalate esters, the intermediate responsible for that peak probably does not contain the phenolic portion of the original reagent. The thermolysis of the hypothetical 1,2-dioxetanedione may have a mechanism similar to the decomposition of dioxetanes. The mechanism for the decomposition of dioxetanes without fluorophor has been studied (17). In the first step a singlet biradical is formed by homolytic cleavage of the 0-0 bond. The formation of a biradical species has been supported by kinetic isotope studies, substitution studies, and thermodynamic calculations (17). Intersystem crossing allows the singlet biradical to become a triplet biradical. Decomposition of the triplet biradical gives an excited triplet ketone that emits light by phosphorescence. Dioxetanes show mostly triplet emission on thermolysis. The thermodynamic parameters calculated for 1,2-dioxetanedione, dioxetanones, and dioxetanes are similar (18). If
ANALYTICAL CHEMISTRY, VOL. 62,NO. 14, JULY 15, 1990 Scheme
1535
I1
v-v
0 0
0 II 0 II
c-c !
I
0-0
l-c
O R fb
dL
t
-
-RR
10 01
c-c
-
1
1
10
or
1,Zdioxetanedione behaves according to the same mechanism proposed for dioxetanes, the decomposition would occur as shown in Scheme 11. Analogous to the emissions observed for dioxetanes, phosphorescence from carbon dioxide may be observed as the emission results from decomposition of 1,2dioxetanedione. Emission from excited COz is not normally observed in the visible region. However, a singlet excited state for bent (122' f 2) carbon dioxide exists about 46 700 cm-' above the ground state. The bent state of C 0 2 was first characterized from examination of the flame emission spectrum of carbon monoxide (19). The emission produces vibrationally excited ground states because the molecule must go from a bent excited state to a linear ground state during fluorescence. The first excited triplet (3B2) is believed to be at about 32000 cm-' above the ground state (14). Phosphorescence from carbon dioxide has been reported to occur in plasmas (14). A multiband spectrum centered around 450 nm is observed (14). If phosphorescence from carbon dioxide is responsible for the 450-nm background emission, then 1,2-dioxetanedione would correspond to intermediate y in Givens' kinetic scheme. Since triplet excited states are effectively quenched by molecular oxygen or the presence of heavy atoms, oxygen or heavy atoms might quench a background emission resulting from excited triplet COP Although dissolved oxygen is always present in the peroxyoxalate system from decomposition of hydrogen peroxide, the blank response increased about 50% when the hydrogen peroxide solution was nitrogen purged. If 5 mM methylene chloride was added to a solution of TCPO (1.3 mM) and hydrogen peroxide (0.32 M), the response was reduced about 20%. The long-wavelength features in the background spectra with peroxyoxalate reagents may arise from chemiluminescent decomposition of the 'fast" intermediate x in Givens' kinetic scheme. Secondary long-wavelength emissions have been reported due to excited dioxetanedione biradicals (14, 20). Since the substitution on the phenolic portion of the peroxyoxalate ester is related to the long-wavelength emission observed, the phenolic residue most likely is contained in both this intermediate and the resulting luminescent byproduct. An excited carbonyl species is a possible light-emitting source. When a fluorophor is the analyte in a peroxyoxalate system, the detection limit is determined by both the fluorescent analyte emission and the background emission. The relationship between the different mechanisms that produce analyte and background emissions may be studied by observing both emissions simultaneously in a single system. If the presence of a fluorophor favors light production by the fluorophor instead of the background pathway, measurement of low levels of fluorophor may not be limited by the background. To investigate this, a fluorophor with emission beyond the 450-nm emission of the background was chosen. A low level of the fluorophor, 16,17-diheptanoyloxyviolanthrone,was added to DNPO and hydrogen peroxide
WAVfitNGTYl"m1
Figure 3. Emission spectrum of DNPO and hydrogen peroxide with 2.4 X 10" M 16,17diheptanoyloxyviolanthrone. The fluorescence spectrum of the DHV is shown In dashed line.
(Figure 3). The background emission at 450nm is clearly visible superimposed on the fluorescence of the DHV. Thus, the background chemiluminescence is competitive with the fluorophor produced chemiluminescence. The slight shift in the maximum wavelength of DHV is probably due to some overlap of the background fluorescent emission. ANALYTICAL IMPLICATIONS Qualitative and quantitative knowledge of the spectral composition and level of the background could be used to further minimize noise in analytical systems. Under excess hydrogen peroxide conditions, one primary intermediate seems to predominate, and the background emission is centered around 450 nm, with little emission at longer wavelength. With fluorophors emitting above 500 nm, a filter could be used to reduce the background emission without reducing the fluorophor emission intensity, thereby giving higher signalto-noise ratios. Detection limits of DHV were evaluated with and without the use of a filter. The detection limit of DHV was 2.8 X lo-' M without a filter and 1.1 X lo-' M with a 500-nm filter. Some analytical problems that may be encountered while using a fluorophor that fluoresces above 500 nm are (1)lower fluorescence quantum yields and (2) the need for red sensitive photomultipliers. Determination of low levels of fluorophor by peroxyoxalate chemiluminescence is limited by competitive reactions. The reaction of analytical interest is usually the bimolecular reaction between an intermediate and a fluorophor analyte. In direct competition to analyte detection is the unimolecular decomposition of intermediate. At sufficiently low analyte concentrations background emission will predominate. If the analytical reagent concentrations of ester and hydrogen peroxide are varied so that hydrogen peroxide is not in excess, then the second intermediate with a longer wavelength emission is formed, and the background spectrum will be dependent on which peroxyoxalate ester is used. LITERATURE CITED Honda, K.; Sekino. J.; Imal, K. Anal. Chem. 1983, 55. 940-943. Kobayashi, S.; Seking, J.; Honda, K.; Imai. K. Anal. Bbchem. 1981, 112, 99-104. Toyoka, T.; Imai, K. J. Chromfogr. 1983, 282, 495-500. Watanabe, Y.; Imai, K. J. Chromafogr. 1984, 309, 279-266. Sigvardson, K.; Birks, J. W. Anal. Chem. 1983, 55, 432-435. Sivardson, K.; Kennish, J. M.; Birks, J. W. Anal. Chem. 1984, 56, 1096-1102. Mann, 0.; Grayeski, M. L. J. Chromatogr. 1987, 386, 149-158. Weinberger. R.; Mannan, C. A.; Cerchio, M.; Grayeski, M. L. J. Chromafogr. 1984, 288, 445-450. Rauhut, M. M.; et al. J. Am. Chem. SOC. 1987, 89, 6515-6522. Catherall, C. L. R.; Palmer, T. F. J. Chem. SOC.,Faraday Trans. 2 , 1984, 80, 823-834. Alvarez, F. J.; Parekh, N. J.; Matusqewski, B.; Givens, R. S.; Higveh, I.: Schowen, R. L. J. Am. Chem. SOC.1888, 108, 6435-6437.
Anal. Chem. 1990, 62, 1536-1542
1536
(12) Mohan, A. G.; Turro, H. J. J. Chem. Educ. 1874, 51, 528. (13) Bollyky, L. J.; Roberts, B. G.; WhRman, R. H.; Lancaster, J. E. J. Org. Chem. 1889, 34, 836-842. (14) Stauff, J.; Jaeschke, W.; Schlogl, G. Z . Phys. Chem. (Munich) 1978, 99,3748. (15) Steintatt. M. F. D. Bull. Soc. Chim. Be&. 1885, 94, 85-86. (16) Kearns. D. I?.Chem. Rev. 1971, 71, 395-427. (17) Rlchardson, W. H.; StiggalCEstbers, D. L. J. Am. Chem. SOC.1982, 104, 4173-4179. (18) Richardson, W. H.; O'Neal, H. E. J. Am. Chem. SOC. 1972, 94, 8665-a668. (19) Dlxon, R. N. Roc. R. Soc.1963, 275a, 431. (20) Capomacchia, A. C.; Jennings, R. N.; Hemingway, S.M.; D'Souza, P.;
Praqaitrakul, W.; Gingle. A. Anal. Chim. Acta 1887. 196, 305-310.
B. Mann M. L. Grayeski* Department of Chemistry Seton Hall University South Orange, New Jersey 07079
RECEIVED for review December 27,1989. Accepted April 23, 1990.
TECHNICAL NOTES Liquid Secondary Ion Mass Spectrometric Analysis of Natural and Recombinant Proteins and Monoclonal Antibody light Chains with Molecular Weights between 16 000 and 25000 Marshall M. Siegel,* Rushung Tsao, Vivian W. Doelling, and Irwin J. Hollander
American Cyanamid Company, Medical Research Division, Lederle Laboratories, Pearl River, New York 10965 INTRODUCTION Recently, Lacey and Keough ( I ) summarized mass spectral data obtained for small proteins with molecular weights greater than 15000. The ionization methods used for these measurements were plasma desorption (PD), electrospray, laser desorption, and liquid-SIMS (secondary ion mass spectrometry). The liquid-SIMS results were based upon the original work of Barber and Green (2)on small model proteins. Barber and Green demonstrated the liquid-SIMS technique by using a cesium ion gun, operating a t 35 kV, and obtained singly as well as multiply charged molecular ions. In more recent work (31, using porcine trypsin (MW 23460) as the model compound, two features of high mass liquid-SIMS spectra were demonstrated, namely, (1)the linear increase in signal with sample size until saturation at 0.6 nM (14 rg) with a signal-to-noise (S/N) ratio of 5 and (2) the decrease in S / N by about a factor of 2.5 times when the sample was adulterated with 100 times molar excess of NaCl(60 nM of NaCl corresponds to 1.4 r g of NaC1). Saturation and adulteration effects have also been observed for low molecular weight compounds (
