JOURNAL OF MASS SPECTROMETRY, VOL. 31, 275-279 (1996)

Use of 2-Hydroxy-1-naphthoic Acid as a Matrix for Mat r ix-assist ed Laser Desor pt ion/Ioniza t ion Mass Spectrometry of Low Molecular Weight Porphyrins and Peptides

Michael G. Bartlett and Kenneth L. Buscb* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA

Christopber A. Wells and Kevin L. %hey Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA

Use of the matrix-assisted laser desorption/ionization matrix 2-hydroxy-1-naphthoic acid provides a 2-4foM enhancement of ion signal for low molecular mass porphyrins over other matrices including a-cyano-4- hydroxycinnamic acid, 2-(4-hydroxyphenylazo)benzoic acid, sinapinic acid and 2,Sdibydroxybenzoic acid. Tetraphenyl-, octaethyl-and tetramethoxyphenylporphyrins give strong signals corresponding to [ M + HI + ions and limits of detection between 100 and 500 fmol. Significant fragmentation is observed for the octaethylporpbyrins at high laser power and moderate fragmentation is observed for the other classes of porphyrins. The 2-hydroxy-l- naphtboic acid matrix also gives strong signals corresponding to IM + H] + and/or [M + Na] + ions for peptides, including metbionine enkephalin, bacitracin, bradykinin, renin substrate and insulin.

KEYWORDS: matrix-assisted laser desorption/ionization; porphyrins; 2-hydroxy-1-naphthoic acid; peptides

INTRODUCTION

Matrix-assisted laser desorption ionization (MALDI)1-3 has had an extensive impact in the analysis of bio- molecules. Typical matrices used in the analysis of bio- molecules include a-cyano-4-hydroxycinnamic acid (HCCA), sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxyphenylazo)benzoic acid (HABA), succinic acid and nicotinic acid.294 These matrices demonstrate efficient absorbance at commonly used laser wavelengths, and form homogeneous microcrystal- line solids with analyte molecules. The most intense signals due to the analyte are obtained from regions of the sample probe where efficient crystallization has o c c ~ r r e d . ~ . ~ Therefore, the ability of the matrix to gen- erate a high-quality crystal is especially desirable.

Porphyrins are a class of molecules that have long been of interest because of their use as geological markers' and photodynamic therapeutic agents.' Except for highly functionalized porphyrins, mass spectra can usually easily be acquired using a heated probe and electron impact (EI) ioni~ation.~ However, thermospray, flow-fast atom bombardment (FAB),'O*' ' chemical ionizati~n,'~-' laser desorption ioniza- tion,"-' ele~trosprayl'~~' and 'W!f plasma des~rption'~ are other ionization methods that have been used to obtain mass spectra of these compounds in

* Author to whom correspondence should be addressed.

a quest for additional structural information or better sensitivity. Recently, MALD120*21 has been used for the analysis of a few porphyrin compounds. One difficulty long associated with the mass spectrometric analysis of porphyrins is the occurrence of reduction processes of the molecular ion that make the determination of correct molecular mass d i f f i ~ U l t . ~ ~ * ~ ~ These reduction processes have been attributed to the addition of hydro- gen across the carbon-carbon double bonds in the porphyrin macrocycle. These hydrogen addition pro- ducts have not yet been observed in the MALDI mass spectra of porphyrins and metalloporphyrins, or in elec- trospray ionization mass spectra, but such reductions are common in both FAB and chemical ionization mass spectra of porphyrins.

The matrix 2-hydroxy-1-naphthoic acid (HNA) has been shown to have many of the properties of successful MALDI matrices, including efficient absorbance at 337 nm (for use of a nitrogen laser), low background matrix ion signals, low matrix adduction and abundant [M + HI' ion generati~n.'~ We show here that the effectiveness of this new matrix is particularly note- worthy for the analysis of low molecular mass porphy- rins, corrins and peptides. Twenty different porphyrin and metalloporphyrin samples (octaethylporphyrins (OEP) Ni(II), Co(II), Mg(II), Fe(III)Cl, Cr(III)Cl, V(IV)O, Cu(II), Ru(1I)CO and Zn(I1); tetra- phenylporphyrins (TPP) Co(II), Fe(III)Cl, Pd(II), Ru(II)CO, Zn(II), Cu(II), Pt(I1) and V(1V)O; tetra- methoxyphenylporphyrins (TMP) Fe(II1)Cl and Co(I1); and pentafluorotetraphenylporphyrin), with molecular

CCC 1076-5174/96/030275-05 0 1996 by John Wiley & Sons, Ltd.

Received 8 September 1995 Accepted 13 November 1995

276 M. G. BARTLETT ET AL.

masses between 500 and 1000 Da, were analyzed by MALDI with the HNA matrix. Additionally, several peptides were analyzed using HNA to assess the sensi- tivity of this matrix for this important class of com- pounds. The peptides ranged in mass from m/z 574 (methionine enkephalin) to m/z 5734 (bovine insulin).

EXPERIMENTAL

MALDI time-of-flight (TOF) mass spectra were acquired on a custom-built TOF instrument that can be operated in both the linear (ion flight path 1.22 m) and reflected (ion flight path 1.98 m) ion mode.24 The TOF mass spectrometer consists of a drift tube, a custom- built ion source and a Chevron microchannel plate detector. A Laser Science VSL-337ND nitrogen laser provides 3 ns pulses of 337 nm light, which are steered and focused so that the 3 x 8 mm beam is reduced to approximately 0.126 mm2 in area. A variable wheel attenuator allows reduction of the laser power to just above the threshold for producing MALDI ion signals. Power densities of 10 MW cm-' are typically used for the MALDI mass spectra presented here. Ions gener- ated from the stainless-steel probe tip (3 mm diameter) are accelerated to 18 keV. The ion current at the Chevron microchannel plate detector (R. M. Jordan Co.) is recorded by a LeCroy 9400A digital oscilloscope with signal-averaging capabilities. Data are then sent to a Zenith 386SX computer, using TOFWARE from ILYS software for storage, manipulation and cali- bration. Mass scale calibration is accomplished using a two-point calibration line established using known masses of a matrix peak and a low molecular mass peptide standard, such as renin substrate, [M + HI+ at m/z 1760. Typically, the ion signals from 20-30 laser shots are summed using the oscilloscope and then trans- ferred to the computer for further data analysis. Instru- mental resolution when operating in the reflected mode is approximately 400 (FWHM). Some digitization is apparent in the data shown in Fig. 3(a) and (b) because of a mismatch between the software dynamic range and that of the digital oscilloscope. The essential features of the data are not affected.

MALDI matrices and peptide samples were pur- chased from Sigma Chemical and used without further purification. Matrices and peptides were dissolved in acetonitrile-water (70 :30, v/v)-0.1% (v/v) trifluoroacetic acid (TFA). Peptide samples were prepared at concen- trations of approximately 10 p ~ , except where noted otherwise. Porphyrin samples were purchased from Midcentury Chemicals or Aldrich Chemical and were used without further purification. Porphyrin samples were dissolved in a 1: 1 mixture of acetonitrile and methanol (20 p~ except for limit of detection studies). All samples were diluted 5 : 1 (v/v) with a 50 mM solu- tion of the chosen matrix, to give a final matrix-to- sample ratio of about 10000: 1. A 1 pl volume of the sample-matrix mixture was applied to the probe and allowed to dry in air. Data shown in the figures and the table reflect best results with usual tuning of laser and instrument parameters, and are therefore valid for com- parative rather than absolute purposes.

RESULTS AND DISCUSSION

The matrix HNA shows a low-intensity background signal in the low-mass region when compared with other MALDI matrices. An ion at m/z 171 represents [M - OH]' or [M + H - H20]+, while the [M + HI' ion itself is seen at m/z 189. [M + Na]' and [M + IS]' ions are also observed, and an ion at m/z 314 has been identified as [2M + H - COOH - H20]+. Such photoinduced loss of the COOH group has been reported for both nicotinic and vanillic acid matrices and has been used to explain protein matrix adduction. In some mass spectra of the HNA matrix, we observe higher mass cluster ions correspond- ing to products of polymerization after loss of the car- boxylic acid group ions (at m/z 287 and 430), but the intensities of these signals are generally small.

The low laser power MALDI spectra of porphyrins using the HNA matrix are generally very simple. Figure 1 shows the positive-ion MALDI spectrum of 500 ng of zinc(I1) OEP. The protonated molecular at m/z 599 is the base peak in the spectrum; the ion at m/z 537 represents loss of the metal atom. At higher resolutions, using the reflector, the isotopes of zinc can be seen in the protonated molecule signal, and ions can be seen that correspond to losses from the side-chain ethyl groups. Most fragmentation reactions for the porphy- rins occur as prompt reactions in the ion source, and are so observed in the linear mass spectral data. Ions formed as a result of metastable decay are observed in the reflected data.14 The rate constants for these meta- stable dissociations are - lo6 s-'. Since the transit times of the ions in this instrument are on the order of 10-20 ps, metastable contributions are often evidenced by shifts in the fragment ion mass assignments of 4-5 mass units in the reflected mode. The mass assignments are correct within 1 mass unit when operating in the linear mode.

When the mass spectrum is acquired at a higher laser power, the amount of fragmentation observed increases. At high laser powers, we observe multiple loss of methyl and/or methylene groups for octaethylporphyrins (OEP), the loss of one phenyl group for tetra- phenylporphyrins (TPP), the loss of a methyl and/or methylene group in the spectra of tetra- methoxyphenylporphyrins (TMP) and the loss of the metal atom for simple metalloporphyrins.

The MALDI mass spectrum for iron(II1) chloride TMP in HNA matrix contains an ion at m/z 825 that corresponds to [M + HI+ for FeCl TMP. It is signifi- cant that the [M + HI+ ion is observed for [M(III)porphyrin] +[XI - type species, since most other ionization methods do not allow the counter ion associ- ated with these types of porphyrins to be determined. An ion at m/z 790 is attributed to the loss of a chlorine atom from the ion at m/z 825, and an ion at m/z 734 is created by the loss of FeC1. Identification of the chlo- ride counter ion was possible for all iron-containing porphyrins. However, the chloride counter ion was not incorporated in ions formed from chromium(II1) chlo- ride OEP. The vanadium(V) oxide porphyrins all show the presence of the oxygen atom, but metalloporphyrins that have carbonyl groups, such as ruthenium(I1) car-

NEW MALDI MATRIX FOR PORPHYRINS AND PEPTIDES 277

ZnOEP

Linear MALDI -- HNA

537

*HI+

599

I I I I r I I m 3w 400 500 600 700 800

mlz

Figure 1. MALDI mass spectrum of zinc(l1) octaethylporphyrin at low laser power.

Table 1. Comparison of the relative abundances of ions derived from various porphyrin and peptide samples with sample preparation in different MALDI matrices

Renin Methionine NiOEPb CoTMPb substrate Bacitracin enkaphalin Insulin

Matrix' [M + H I + [M + H I + [M + H I + [M + H]* [M + Na]' [M + HI+ [M + Na]+ [M + HI'

SA 0.86 0.47 0.67 0.08 0.00 0.07 0.01 1.25 DHB 1 .oo 1 .oo 1 .oo 1 .oo 0.20 1 .oo 0.01 1 .oo HCCA 0.36 0.67 0.33 1.30 0.08 0.81 0.08 1.50 H NA 4.29 1.94 1.67 0.63 0.20 0.35 0.70 0.42 HABA 0.32 0.44 ND' 0.03 0.1 5 0.02 0.25 0.08

* SA - Sinapinic acid; DHB = 2.5-dihydroxybenzoic acid; HCCA - a-cyano-4-hydroxycinnamic acid; HNA - 2-hydroxy-I -naphthoic acid; and HABA - 2-(4-hydroxyphenylazo)benzoic acid.

' ND is not determined. OEP = octaethylporphyrin; TMP - tetramethoxyphenylporphyrin.

CO(ll) OEP YALDI - HNA Reflector fM+H]+

593

540 580 600

r n l z

Figure 2. MALDI mass spectrum of 140pg of cobalt(l1) octaethylporphyrin.

278 M. G. BARTLETT ET AL.

[M+H1+ 1061

Brrdyklnln

Llnoar YALDI--CA

k I I 1 I I I 1

m loo0 law 1- 1Sm lam ama m/z

rnl2

Figure 3. MALDI mass spectra of bradykinin in (a) a-cyano-4-hydroxycinnamic acid and (b) 2-hydroxy-I -naphthoic acid

bony1 TPP, do not retain that group in the MALDI factor of 2-4) for porphyrin analysis than other spectra obtained with the HNA matrix.

A direct comparison of the intensities of the The mass spectral sensitivity achieved with HNA is [M + HIt ions formed from equal amounts of generally better than values reported in the literature nickel(I1) OEP and cobalt(I1) TMP analyzed in five for other matrices. In the work of Hercules and co- common MALDI matrices is given in Table 1. The workers,” the porphyrin sample was dissolved in nitro- HNA matrix produces higher signal intensities (by a phenyloctylether and then applied to a probe that had

common matrices such as HCCA, HABA, SA or DHB.

been coated with nitrocellulose. Using this combination, they reports that a limit of detection of 500 pg was achieved for Zn(I1)TPP. A similar limit of detection for Zn(I1)TPP was found using HNA as a matrix (420 pg). Several of the porphyrins could be analyzed from HNA matrices with significantly improved limits of detection. An example is provided in Fig. 2, which establishes a limit of detection (using a signal-to-noise ratio of 3 : 1) of 140 pg for Co(1I)OEP when using HNA as the matrix and measuring the abundance of the [M + H]+ ion. The ions at m/z 574 and 556 in the mass spectrum in Fig. 2 are low-intensity signals from the matrix itself.

HNA has also been effective for the analysis of pep- tides weighing less than 2000 and a-cyano-4-hydroxy- cinnamic Da. In general experience, we find DHB to work well for molecules up to masses around 10,000 Daltons. SA does not produce as intense a signal as DHB or HCCA for low-mass molecules, but provides more intense signals for molecules of mass above 10000 Da. In our experience, HABA is not as useful as other matrices for molecules below 20000 Da in mass. Inten- sities of the [M + H] ' signals from methionine enkep- halin (m/z 574), the cyclic peptide bacitracin (m/z 1423), renin substrate (m/z 1760) and bovine insulin (m/z 5734) using these different MALDI matrices and HNA are summarized in Table 1. HNA shows intermediate sensi- tivity for the [M + HI+ ion from methionine enkepha- lin and bacitracin compared with other matrices, and increased signal strength for the [M + HIf ion from renin substrate. A comparison of HNA with HCCA for measurement of the mass spectrum of bradykinin is shown in Fig. 3, where 59 amol of samples were spotted with each matrix. The spectra show excellent signal-to- noise ratios, and a 2.8-fold increase in signal with use of HNA rather than HCCA. One interesting observation is the increased abundance of [M + Na]' and [M + K]' ions in the mass spectra of peptides when using HNA as a matrix. A comparison of the [M + Na]' ion from methionine enkephalin and bacitracin shows that HNA

has substantially better response for the sodiated ions than any other matrix tested. This phenomenon is a topic of further investigation.

CONCLUSIONS

NEW MALDI MATRIX FOR PORPHYRINS AND PEF'TIDES 219

REFERENCES

The use of the HNA matrix gives increased sensitivity for the molecular ion of low molecular mass porphyrins in MALDI, and when high laser power is used, a large degree of fragmentation is observed. Detection limits for porphyrins are in the low picomole to high femtomole range. For the analysis of peptides, HNA gives good sensitivity for molecules under 2000 Da in mass. The increased abundance of ions associated with the addi- tion of alkali metals may demonstrate a need for salt removal before using the HNA matrix with such samples, although the addition of a known amount of salts might allow for a more positive determination of molecular mass via concurrent [M + HI', [M + Na]+ and [M + K] + ion formation.

The limit of detection for the porphyrins in HNA is 100-500 pg of porphyrin sample on the probe. HNA compares favorably with other matrices for the analysis of peptides up to mass 2000 Da, but does not provide more intense signals for higher mass peptides. Improved low-mass sensitivity obtained using the HNA matrix (better signal-to-noise characteristics) may be significant in the analysis of important low molecular mass com- pounds found in cells and tissues such as pep tide^,'^ exogenous pharmaceuticals and their metabolites or toxins.'

Acknowledgements

K.L.S. and C.A.W. acknowledge support from the Medical University of South Carolina Research Funds. M.G.B. and K.L.B. acknowledge support from Chevron Research Corporation and from Unilever. The authors also thank Dr P. Privitera for the generous gift of the brady- kinin sample.

1. 2.

3. 4.

5.

6.

7.

8.

9.

10.

11. 12.

M. Karas and F. Hillenkamp,Anal. Chem. 60,2299 (1988). F. Hillenkamp, M. Karas, R. C. Beavis and B. T. Chait, Anal. Chem. 63,1193A (1 991 ) . R . J. Cotter,Anal. Chem. 64,1027A (1992). P. Juhasz, C. E. Costello and K. Biemann, J. Am. SOC. Mass Spectrom. 4,399 (1 993). K. Strupat, M. Karas and F. Hillenkamp, lnt. J. Mass Specrrom. Ion Processes 111,89 (1991). F. Xiang and R. C. Beavis, Rapid Commun. Mass Spectrom. 8, 199 (1994). E. J. Gallegos and P. Sundaraman, Mass Spectrom. Rev. 4, 55 (1985). F.-Y. Shiau, P. A. Liddell, M. G. M. Vicente, N. V. Ramana, K. Ramachandran, S.-J. H. Lee, R . K. Pandey, T. J. Dougherty and K. M. Smith, Future Direct. Appl. Photodyn. Ther. 6, C. J. Gomer (ed.), SPlE Optical Engineering Press, Bellingham, WA, 71 (1990). K. M. Smith, in Porphyrins and Metalloporphyrins. edited by K. M. Smith. Elsevier, Amsterdam, 382 (1975). M. Barber, R . S. Bordoli, G. J. Elliott, R . D. Sedgwick and A. N. Tyler, Anal. Chem. 54,645A (1 982). E. K. Fukuda and J. E. Campana,Anal. Chem. 57,949 (1 985). G. J. Shaw, G. Eglinton and M. E. Quirke, Anal. Chem. 53, 2014 (1981).

13.

14.

15.

16. 17.

18.

19.

20.

21.

22. 23.

24.

B. Tolf, X. Y. Jiang, A. Wegrnann-Srente, L. A. Kehres, E. Bunnenberg and C. Djerassi, J. Am. Chem. SOC. 108, 1363 (1 986). J. C. Tabet, M. Jablonski, R . J. Cotter and J. E. Hunt, lnt. J. Mass Spectrom. /on Processes 65, 1 05 (1 985). R . S. Brown and C. L. Wilkins, J. Am. Chem. SOC. 108. 2447 (1 986). R . S. Brown and C. L. Wilkins,Anal. Chem. 58,3196 (1986). G. J. Van Berkel, S. A. McLuckeyand G. L. Glish,Anal. Chem. 63, 1098 (1991). R. D. Smith, J . A. Loo, C. G. Edmonds, C. J. Barinaga and H. R . Udseth,Anal. Chem. 62,882 (1 990). J. E. Hunt, R . D. Macfarlane. J. J. Katz and R. C. Dougherty. J. Am. Chem. SOC. 103,6775 (1 981 ). J. Ha, J. D. Hogan and D. A. Laude, Jr, J. Am. SOC. Mass Spectrom. 4, 159 (1 993). Y. Kim, S. Zhao, A. G. Sharkey and D. M. Hercules, in Pro- ceedings of the 40th ASMS Conference on Mass Spectrom- etry and Allied Topics, p. 322 (1 992). H. H. Schurz and K. L. Busch, Energy Fuels 4, 730 (1990). H H Schurz and K. L. Busch. Org. Mass Spectrom. 27, 59 (1992). M. G. Bartlett, C. A. Wells, K. L. Schey and K. L. Busch, in Pro- ceedings o f the 45th Pittsburgh Conference, p. 462 (1994).