The Use of Osazones as Matrices for the Matrix-Assisted Laser Desorption/Ionization Mass...
Transcript of The Use of Osazones as Matrices for the Matrix-Assisted Laser Desorption/Ionization Mass...
ANALYTICAL BIOCHEMISTRY 244, 144–151 (1997)ARTICLE NO. AB969874
The Use of Osazones as Matrices for the Matrix-AssistedLaser Desorption/Ionization Mass Spectrometryof Carbohydrates
Peng Chen, Andrew G. Baker, and Milos V. Novotny1
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Received June 10, 1996
nic acid (11), a-cyano-4-hydroxycinnamic acid (12), 2-Fifteen sugar osazones were synthesized and evalu- aminobenzoic acid (13), 3-hydroxypicolinic acid (14), 3-
ated for their use as matrix-assisted laser desorption/ aminoquinoline (3-AQ) (15), 2,5-dihydroxybenzoic acidionization matrices. Various carbohydrate samples were (DHB) (16), and DHB/additive comatrices, includinganalyzed, indicating that D- or L-arabinosazone, pre- ‘‘super DHB’’ (with 2-hydroxy-5-methoxybenzoic acidpared from D- or L-arabinose and phenylhydrazine, gave as an additive) (17) and DHB/HIQ (with 1-hydroxyiso-the overall best performance. The arabinosazones facili- quinoline as an additive) (18).tated superior analyses in providing a highly uniform Osazones, the products of the reaction between a car-distribution of sample molecules within the matrix mi- bohydrate and an aromatic hydrazine, are among thecrocrystals, and a possibility of using low laser energy best examples of crystallinity in organic chemistry.for irradiation, leading to high spectral resolution and Synthesized first in 1884 by Emil Fischer as a preludelow matrix background. The arabinosazones with a mod- to his remarkable studies on carbohydrate stereochem-erate content of sodium ions promoted specific fragmen-
istry, osazones were used, for some time, in a micro-tation for certain carbohydrates tested. The 50 fmolscopic proof of carbohydrate structures.amounts of maltohexaose and maltoheptaose (present in
During a search for suitable MALDI matrices for the0.1 ml of sample/matrix solution) were detected at a sig-carbohydrate analysis, we became interested in osa-nal-to-background ratio of 3:1. q 1997 Academic Press, Inc.zones because of their distinct microcrystallinity, aro-maticity with an appropriate UV absorption at 337 nm,their nearly neutral character, high surface adhesionto the stainless-steel sample plates, and their potentialA proper choice of the sample matrix has been crucial (through a facile structural modification in either theto success in the matrix-assisted laser desorption/ion- sugar type or the aromatic moiety) for elucidating theization (MALDI)2 mass spectrometry of various biomol- elusive nature of the MALDI process. We wish to reportecules (1, 2). A relatively high tolerance of the MALDI here the preliminary results with arabinosazone, somethod for excessive amounts of biochemical buffers, far the best osazone matrix that we have identified, insalts, and impurities makes this technique particularly carbohydrate analysis. A comparison has been madeattractive for investigating minute quantities of bio- with DHB which appears to be a popular choice in themolecules isolated from complex biological materials analysis of glycoconjugates (2, 19–22).(1, 3, 4). In spite of the enormous practical impact of
this method, the role of different matrices in theEXPERIMENTALMALDI process remains poorly understood. Most seem
Instrumentation. A Voyager Linear BioSpectrome-to have been chosen empirically, while a search for thetry Workstation MALDI/time-of-flight mass spectrome-optimal MALDI media for new applications continuester from Perseptive Biosystems (Framingham, MA)(5–10). The most widely used matrices include sinapi-was used in all measurements, except the result in Fig.4E that was obtained on a Voyager DE-RP model from1 To whom correspondence should be addressed. the same company. The system utilizes a pulsed nitro-2 Abbreviations used: MALDI, matrix-assisted laser desorption/gen laser, emitting at 337 nm. The extraction voltageionization; 3-AQ, 3-aminoquinoline; DHB, 2,5-dihydroxybenzoic acid;
HIQ, 1-hydroxyisoquinoline. used was 28 kV, except for Fig. 4E where 20 kV were
144 0003-2697/97 $25.00Copyright q 1997 by Academic Press, Inc.
All rights of reproduction in any form reserved.
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ARABINOSAZONE AS SPECTROMETRIC MATRIX FOR CARBOHYDRATES 145
applied. The mass spectrometer was employed exclu- they are referred to as ‘‘sodium-rich osazones’’ in thefollowing text. Melting point of the sodium-rich D-ara-sively in the positive ion mode. Pressure in the ion
source was maintained between 2.0 1 1007 and 1.0 binosazone was determined as 1667C. Its NMR spectrawere characteristic, indicating a high purity following1 1006 Torr. For the laminarin analyses, low-mass-
rejection of 0–1000 m/z was used to exclude most of its facile preparation. 1H NMR (DMSO-d6 , 400 MHZ):d 3.59 (m, 2H), 4.12 (m, 1H), 4.47 (t, 1H), 4.64 (d, 1H),the ions with m/z ratio less than 1000. All spectra were
smoothed at 19 points (the Savitsky–Golay procedure) 5.37 (d, 1H), 6.8–7.4 (10 ArH), 7.77 (s, 1H); 13C NMR(DMSO-d6 , 400 MHZ): 64.1, 74.7, 74.9, 112.4, 113.2,with order of 2, except the case in Fig. 4E where the
delayed extraction and reflector modes were applied. 120.3, 120.7, 130.0, 130.1, 134.7, 137.5, 144.6, 145.0. Tofurther remove the sodium ions from the preparation, aThe laser output energy values were measured using
a Model RM 6600 Universal Radiometer with a Model 60% ethanol/aqueous solution was first heated to boil-ing and then slowly added to the sodium-rich arabino-RJP-735 probe from Laser Precision Corp. The NMRsazone under magnetic stirring until all precipitate justmeasurements were carried out on a Varian VXR400dissolved. A subsequent cooling at room temperaturespectrometer. The melting point of arabinosazone wasresulted in crystallization. The microcrystals were col-measured using a Thomas Hoover capillary meltinglected through filtration. After another twofold recrys-point apparatus (Arthur H. Thomas Co., Philadelphia).tallization and a final removal of solvent in vacuo, aMaterials. D-Erythrulose, D-ribose, D- and L-ara-brownish osazone was obtained. The osazones of thisbinoses, D-xylose, D-lyxose, D-allose, D-glucose, D-man-type are referred to as ‘‘sodium-free osazones’’ in thenose, D-galactose, a-D-rhamnose, L-sorbose, maltose,text. The arabinosazone matrices appear to be stableb-lactose, a-D-melibiose, laminarin (from Laminaria dig-at room temperature, but storage under 47C is recom-itata), maltotriose, maltotetraose, maltopentaose, malto-mended as a precaution.hexaose, maltoheptaose, ribonuclease B (type III-B from
Sample preparation for MALDI. A matrix bulk so-bovine pancreas), DOWEX-50W (H/) resin, and DOWEXlution was prepared by dissolving 1 mg osazone (or1 (Cl0) resin (exchanged to OH0 type with NaOH) wereDHB, for comparison) in 50–200 ml ethanol or metha-purchased from Sigma. 2,5-Dihydroxy-benzoic acid,nol. A 0.8- to 1.2-ml aliquot of this solution was pre-5-methoxysalicylic acid, 1-hydroxy-isoquinoline (alsomixed with 1.0 ml aqueous sample solution (with a typi-known as isocarbostyril), 3-aminoquinoline, phenyl hy-cal analyte concentration ranging from 1 to 100 pmol/drazine hydrochloride, and sodium acetate were obtainedml). The final solution was subsequently spotted on afrom Aldrich. The Flavobacterium meningosepticum N-polished stainless-steel sample plate and allowed toglycosidase F (a recombinant product from Escherichiadry. The on-plate mixing of 0.05 ml of sample solutioncoli) was received from Boehringer-Mannheim as a 20-with 0.05 ml of matrix solution was achieved throughunit solution containing 50 mM sodium phosphate (pHthe use of 1-ml scale Hamilton syringes. When DHB7.2), 12.5 mM EDTA, and 50% glycerol.was used as a matrix, it was often necessary to perform
Preparation of osazones. The osazones were pre- recrystallization on the plate with ethanol to improvepared from phenylhydrazine and 15 different sugars the laser desorption process.according to the previously published procedures (23).
Enzymatic cleavage of carbohydrates from glycopro-Briefly, 2.0 mmol sugar, 4.0 mmol (0.58 g) phenylhy-tein. Ribonuclease B (1.5 mg) was dissolved in 150 mldrazine hydrochloride, and 10 mmol (0.82 g) sodium buffer solution (20 mM sodium phosphate and 40 mMacetate were placed in a 25-ml test tube, followed by EDTA, pH 7.95) before addition of 7.5 ml (1.5 units ofan addition of 6 ml distilled water. The test tube was activity) of N-glycosidase F. The reaction mixture wassubsequently heated in a boiling-water bath, with an incubated at 377C for 12 h. The released carbohydratesoccasional stirring. The time needed for the formation were desalted through a self-made fritted plastic sy-of osazone yellow precipitate varies, depending on the ringe packed with 2.5 ml mixed resin of DOWEX -50W
sugar type. After the initial formation of a yellow pre- (H/) and DOWEX 1 (OH0). The salt-free carbohydratescipitate (sometimes a brownish oil) (23), the test tube were further isolated from the protein by passagewas kept in boiling water for another 20 min, and then through a C18 Sep-Pak cartridge (Waters Corp., Mil-cooled down. The characteristic yellow precipitates of ford, MA) and concentrated to about 30-ml volume un-osazones were recovered through centrifugation and der a stream of nitrogen, at 257C.decanting. The precipitates were washed with 10 mldistilled water under shaking and were recovered
RESULTS AND DISCUSSIONagain through centrifugation and decanting. After athreefold wash, the precipitates were further filtered We have prepared osazones from a number of four-,to remove residual water and dried in vacuo. The osa- five-, and six-carbon monosaccharides and evaluatedzones prepared in such a manner (with a typical yield them briefly for suitability as MALDI matrices. We
were particularly interested in a matrix that offers aover 65%) still had a substantial amount of sodium;
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minimum background spectrum, minimum adduct for- sample/matrix preparation. In general, among all thematrices tested for various carbohydrate samples, ara-mation with sample molecules, and good sample de-
sorption at a relatively low laser power. While most binosazones offered consistently a more uniform sam-ple/matrix distribution and a lower laser energy neededosazones exhibited a fine crystalline formation on the
plate surface, their desorption characteristics differed. for desorption. In addition, it also featured the overallbest spectral resolution, the lowest detection limits,For example, while the osazones made from typical six-
carbon sugars (D-allose, D-glucose, D-mannose, D-galac- and good tolerance for a salt-containing buffer. For thesake of simplicity, only DHB is reported for comparisontose, L-sorbose, and a-L-rhamnose) performed reason-
ably as matrices, their solubilities in aqueous solutions in this study.Figure 1 compares the use of arabinosazone and(where carbohydrate samples are typically handled)
were generally less than those made from the pentoses. DHB as matrices in the analysis of laminarin, a D-glucan featuring 1–3 and 1–6 linkages. With both ma-Among the pentoses tested in this work (D-ribose, D-
and L-arabinose, D-xylose, and D-lyxose), arabinose (in trices, the individual oligosaccharide peaks appear assodium adducts. The sodium content in the sample/either D- or L-configuration) gave the best results in
desorption experiments. A four-carbon monosaccharide arabinosazone cocrystalline matrix is reflected in theratio of peaks at 329.38 [M / 1]/ and 351.36 [M /Na]/D-erythrulose performed acceptably for small peptides,
but showed little value in carbohydrate analysis. The (M refers to the average mass of the arabinosazone).A high sodium content in the sample/arabinosazoneosazones prepared from three disaccharides (maltose,
b-lactose, and a-D-melibiose) featured good solubilities, cocrystalline medium results in a dominating [M /Na]/ in the matrix background peaks, while a low so-but inferior desorption behavior.
Similarly to the 3-AQ matrix, the arabinosazones do dium content gave a much less intense [M / Na]/ buta substantially increased [M / 1]/. Compared to DHB,not have any carboxylic acid group. The arabinosazone
aqueous–alcohol solutions that we prepared had typi- a lower matrix background and a better mass resolu-tion are readily observed with the osazone. The D- orcal pH values around 5.5 and could be partially neutral-
ized with sodium hydroxide, while DHB in the same L-arabinosazone, when deposited on the MALDI platesurface, showed substantially different properties fromaqueous–alcohol solution gave a pH value around 2.0.
Neutralization of DHB with sodium hydroxide gave in- DHB. They generally gave finer microcrystals and amore uniform distribution that resulted in an excellentferior results. It is potentially beneficial to use milder
matrix solutions because some structural changes in spot-to-spot signal reproducibility. The less-intense la-ser irradiation needed for these new matrices seemedsamples could occur in the acidic media. For instance,
the glycans containing sialic acid may undergo desialy- to decrease the energy dispersion during the desorp-tion/ionization process, causing a better spectral reso-lation in the low-pH solution (24). Incidentally, an at-
tempt to acidify the sample/arabinosazone matrix solu- lution and a lower matrix background. The oligomericenvelope observed in Fig. 1B, favoring higher m/z, istion led to a color change from yellow to red
(presumably, due to an acid hydrolysis of the hydra- more representative (25) of the sample compositionthan that observed with DHB (Fig. 1A). Presumably,zone), and inferior detection.
We wish to communicate the results obtained with the higher laser power used with DHB caused someunwanted fragmentation.D-arabinosazone, the structure of which is given below:
The sodium-rich and sodium-free arabinosazoneshad different appearance after their preparation: theformer had a yellow, fluffy look, while the latter pro-duced more compact, brownish aggregates. Their dif-ferent appearance in the solid state seems to be relatedto the sodium content. Figure 2 compares the MALDIspectra of several maltoses obtained under differentexperimental conditions: (A) DHB; (B) sodium-free ara-binosazone; and (C) sodium-rich arabinosazone. In allinstances, the maltoses with 3–7 oligomeric units arefound at the expected masses for their sodium adducts.The sodium-free osazone matrix (B) and DHB (A) alsoyield minor adduct peaks at [M / K]/, but no suchFor a comparison, a number of other known matrices
were tested, which included ‘‘super DHB,’’ DHB/HIQ, peaks are observed with the sodium-rich osazone (pre-sumably, due to the presence of a large excess of sodiumand 3-AQ. In our hands, the observed properties of
these matrices (relative to the DHB) varied with carbo- ions). Instead, major fragments at [(M / Na)-98] andsome minor fragments at [(M/Na)-98/Na] and [(M/hydrate samples of different types, or samples of the
same type but with different chain lengths, and the Na)-98 / K], etc. (not labeled in Fig. 2C) were observed
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FIG. 1. Mass spectra of laminarin in (A) DHB matrix, recrystallized on the plate with ethanol, and (B) sodium-rich D-arabinosazone. A14.1 mJ/pulse laser energy was used for (A) and 9.2 mJ/pulse for (B). The m/z differences between adjacent sugar peaks are 162, correspondingto the expected addition of one glucose monomer unit. (M / 1) and (M / Na) are the protonated and sodiated arabinosazone molecules,respectively.
using laser energies ranging from 6.5 to 12 mJ/pulse. (Fig. 3) at m/z 1258, 1420, 1583, and 1745, which is inagreement with the previously reported data (20, 21).In fact, further breakdown of these fragments, with
differences from one to several monomer units, was Some minor peaks were also reproducibly observedwith either sodium-free and sodium-rich arabinosa-also noted when only maltoheptaose was used (data
not shown). Such fragmentation was also observed with zone, suggesting additional, minor carbohydrate at-tachments. Once again, compared to the DHB matrix,unrecrystallized 3-AQ, but not when the sodiated DHB
was tested for comparison (data not shown). To our the use of arabinosazone facilitated a lower laser powerand provided better mass-spectral resolution (compari-knowledge, this type of fragmentation is uncommon.
Its mechanism is currently under study, with a poten- son not shown).The good performance of arabinosazones with carbo-tial application for carbohydrate sequencing. It should
be noted that the presence of this fragmentation was hydrate sample could also be attributed to its multihy-droxy character, which is comparable with the ana-somewhat dependent on a sugar type, which is also
a phenomenon deserving additional investigation. For lyzed sugar molecules. Such structural compatibilitymay favor formation of sample/matrix cocrystals. Theinstance, such fragmentation was also observed for de-
xtrin samples (data not shown), but not for laminarin microcrystals were distributed uniformly on the sam-ple plate after solvent evaporation without the com-(a D-glucan with predominant 1–3 and 1–6 linkages).
The sugars with 1–4 linkages appear to have a greater monly noticed ‘‘sweet spot’’ phenomenon (8, 26). Unlikewith DHB, the on-plate recrystallization was unneces-tendency for this type of fragmentation.
The major oligosaccharides cleaved enzymatically sary. Incidentally, due to the low solubility of the arabi-nosazone in pure water, an on-plate wash with waterfrom ribonuclease B were detected as sodium adducts
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FIG. 2. Mass spectra of the carbohydrate mixture containing equal weights of maltotriose (Mr 504.4), maltotetraose (Mr 666.6), maltopen-taose (Mr 828.7), maltohexaose (Mr 990.9), and maltoheptaose (Mr 1153.0) in (A) DHB matrix, (B) sodium-free D-arabinosazone, and (C)sodium-rich D-arabinosazone. A 12.5 mJ/pulse laser energy was used for A, 7.2 mJ/pulse for B, and 6.8 mJ/pulse for C. Peaks a through ecorrespond to the sodiated maltotriose to maltoheptaose, respectively; peaks f and g signify the potassiated maltohexaose and maltoheptaose;peaks h through k represent fragmentation from sodiated maltotetraose to maltoheptaose, with the overall losses of 98 mass units,respectively.
can help removal of undesirable water-soluble impuri- salt-containing samples. Figure 4 depicts the usefulnessof the matrix for sodium-containing samples, using theties, such as salt crystals and surfactants, present with
the sample/matrix aggregates; as a result, better detec- sodium-free D-arabinosazone in the analysis of maltohex-aose and maltoheptaose. Figure 4A shows the analysistion of the analytes may be achieved. In some cases, a
further on-plate recrystallization with a 1:1 volume ra- of 10 pmol each of maltohexaose and maltoheptaose ina 4.5-ml matrix solution containing 15-nmol sodium-freetio of ethanol and water (after the water wash) may
be helpful. Additionally, the use of potassium acetate arabinosazone. Inclusion of 10 and 100 pmol sodium hy-droxide, respectively (Figs. 4B and 4C), resulted in im-(instead of sodium acetate) in the arabinosazone prepa-
ration led to potassium-containing arabinosazone; as proved spectral resolution and a higher ion production,presumably because the sodium ions assisted either for-a result, dominant potassium adducts with the sugar
molecules could be observed. mation of the sample/matrix cocrystals or the desorption/ionization process itself. However, larger amounts of so-It is extremely important to note that the use of so-
dium-rich arabinosazone did not cause any significant dium (1 nmol) caused an increase in the detection limit,as shown in Fig. 4D. It should be noted that the fragmen-deterioration in the spectral quality, indicating that when
a sodium-free arabinosazone is used, the matrix solution tation was more frequently seen with a higher contentof sugar molecules and sodium ions; therefore, the above-can tolerate a certain amount of sodium content in the
sample. The MALDI/MS analysis of biologically im- mentioned fragmentation was not observed in Fig. 4D.In order to detect smaller amounts of analytes than thoseportant glycans, which are usually available in only mi-
nute quantities and in buffer solutions, must tolerate shown in Figs. 4B and 4C, an on-plate mixing of 0.05 ml
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ARABINOSAZONE AS SPECTROMETRIC MATRIX FOR CARBOHYDRATES 149
FIG. 3. Mass spectrum of the N-glycans released from ribonuclease B. The sodium-free D-arabinosazone was used as a matrix. A 8.8 mJ/pulse laser energy was applied.
of sample solution (with a sugar concentration of 1 pmol/ matrix through the use of nitro- and dinitrophenylhy-drazine during the osazone preparation were not suc-ml each) and 0.05 ml of matrix solution was carried out.
A lower detection limit (down to 50 fmol at a signal- cessful. The formed derivatives of arabinose and ga-lactose could be used as matrices, but the desorptionto-background ratio of 3:1, with no data smoothing) for
maltohexaose and maltoheptaose was achieved, as de- process necessitated significantly greater laser powerthan experienced with the arabinosazones. However,picted in Fig. 4E. Due to the use of the delayed extraction
and the reflector technology in this case, the isotopic it is conceivable that a proper combination of espe-cially designed hydrazines and modified sugars couldpeaks can be visualized.
Although other matrices are available for the MALDI/ meet additional applications in MALDI mass spec-trometry.mass spectrometry of small peptides, we briefly investi-
gated the applicability of arabinosazone for such com- In conclusion, the osazone formed from the reactionof phenylhydrazine and arabinose is highly suitablepounds. Using angiotensins as model compounds, we
observed that the sodium-free osazone yielded domi- as a MALDI matrix for the analysis of oligosaccha-rides. Its microcrystalline nature aids in formationnant [M / 1]/ ions (data not shown), the same as seen
with DHB. However, the sodium-rich arabinosazone of a highly uniform layer on the sample plate. Inclu-sion of a small amount of sodium ions in the arabino-matrix desorbed peptides as clusters of sodium ad-
ducts, while the oligosaccharides were still detected as sazone matrix improved the detection limit, while asomewhat larger amount of sodium ions promotedsingle sodium adducts (data not shown). This differen-
tial behavior could be utilized analytically in distin- the specific fragmentation of certain carbohydrates.Sample desorption with this matrix occurs at low la-guishing oligosaccharides from peptides or glycopep-
tides in unpurified cleavage products. ser irradiation, resulting in high mass resolution andlow matrix background.Our attempts at optimizing further the osazone
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FIG. 4. Sodium effects on the spectral resolution and detection limits. Four 4.5-ml sample/matrix solutions were prepared, containing 10pmol each of maltohexaose and maltoheptaose, 15 nmol sodium-free D-arabinosazone matrix, and (A) no NaOH addition, (B) 10 pmol NaOH,(C) 100 pmol NaOH, and (D) 1 nmol NaOH. In E, 50 fmol each of maltohexaose and maltoheptaose was sampled through an on-plate mixingof 0.05 ml of sample solution and 0.05 ml of sodium-free D-arabinosazone matrix solution, with an added sodium content equivalent to B. A13 mJ/pulse laser energy was used in A–D and 5 mJ/pulse in E. 1, sodium adduct of maltohexaose; 2, sodium adduct of maltoheptaose.
9. Woods, A. S., Buchsbaum, J. C., Worrall, T. A., Berg, J. M., andACKNOWLEDGMENTCotter, R. J. (1995) Anal. Chem. 67, 4462–4465.
This research was supported by Grant 24349 from the National 10. Tang, X., Callahan, J. H., Zhou, P., and Vertes, A. (1995) Anal.Institute of General Medical Sciences, U.S. Department of Health Chem. 67, 4542–4548.and Human Services.
11. Beavis, R. C., and Chait, B. T. (1989) Rapid Commun. MassSpectrom. 3, 432–435.
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