JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 34, 1205–1207 (1999)

JMS Letters

Dear Sir,

Influence of Sample Preparation Methodology on theReduction of Peptide Matrix-assisted LaserDesorption/Ionization Ion Signals by Surface–PeptideBinding

In previous work we have shown that polymer surface–peptidebinding has a strong, adverse influence on peptide matrix-assistedlaser desorption/ionization (MALDI) ion signals.1 Our studieshave shown that as the surface–peptide binding affinity increasesthe peptide MALDI ion signal decreases, presumably owing topoor incorporation of the surface-bound peptide in the MALDImatrix. These results were obtained under conditions where thepeptide was first deposited on the polymer surface from anaqueous, phosphate-buffered saline solution and allowed¾45 minfor complete evaporation of the water solvent. Subsequently,small volumes of the MALDI matrix in methanol and 10%trifluoroacetic acid in water were deposited on the peptide-coatedpolymer surface and allowed to dry. In our original report wespeculated that this method of sample preparation might tend toenhance the influence of surface–peptide binding interactions dueto the lengthy drying period of the first step during which timethe peptide has ample opportunity to sample the polymer surfaceand occupy regions of high binding affinity.

The described method of sample preparation is analogous tothat employed in the MALDI mass analysis of peptides whichhave been separated by gel electrophoresis and deposited or elec-troblotted on to polymer surfaces.2 Based on our earlier results,surface–peptide binding effects would clearly be expected to havean impact on the sensitivity of this approach. It is certainly con-ceivable, however, that other methods of sample preparation willalso reflect an effect of surface–peptide binding affinity on pep-tide MALDI ion signals. Furthermore, these effects would not beexpected to be limited to a unique class of polymer surfaces sincemany surfaces, including siliconized surfaces,3,4 metals,5–7 andpolymers8–11 have relatively high binding affinities for numerouspeptides and proteins.

In the present studies we have examined the impact of themethod of sample preparation on the observed inverse rela-tionship between surface–peptide binding affinity and peptideMALDI ion signal intensity using four different sample prepa-ration methodologies. The four methodologies employed includethe method used in our previous studies (described above) asa control, conventional matrix and peptide co-deposition meth-ods and a matrix first followed by peptide deposition method.These approaches were selected to reflect common MALDI sam-ple preparation techniques12 and in an effort to minimize progres-sively the time available for peptide binding and/or peptide accessto surface–peptide binding sites. Clearly, if surface–peptide bind-ing adversely effects peptide MALDI ion signals under these moreconventional sample preparation conditions, this result would haveimportant implications for the broader application of MALDI massspectrometry.

Surfaces with varying peptide binding affinities were preparedby depositing amine-rich films on poly(ethylene terephthalate)

* Correspondence to: G. R. Kinsel, Department of Chemistry andBiochemistry, University of Texas Arlington, Arlington, Texas 76019-0065, USA.E-mail: kinsel@uta.edu

Contract/grant sponsor: NSF; Contract/grant number: BES-9812708;Contract/grant number: CHE-9876249.

Contract/grant sponsor: Texas Higher Education CoordinatingBoard—Advanced Technology Program;Contract/grant number:003656-137.

(PET) (Goodfellow Corporation) polymer substrates using vari-able dury cycle pulsed r.f. plasma deposition13–15 of allyl amine.Disks of 4.8 mm diameter of the PET were cut from 0.125 mmthick sheets of the polymer, cleaned with acetone and securedto glass slides using double-sided tape. The PET disk-containingslides were placed in the r.f. plasma reactor and cleaned furtherusing an argon plasma etch. Subsequently, allylamine was intro-duced to the chamber and the plasma was pulsed on and off in dutycycles of either 3/5, 3/15 or 3/45 ms at 200 W. Previous studieshave shown that these changes in the pulsed r.f. plasma modifi-cation duty cycle result in significant changes in the retention ofthe amine functionality on the surface of the deposited film.16,17

Specifically, as the duty cycle is changed from 3/5 to 3/15 to3/45 ms an increase in retention of amine functionality results.Furthermore, previous studies have shown that, for a variety ofpeptides and proteins, as the surface density of the amine function-ality increases, the surface–peptide binding affinity also increasessignificantly.1 This increase in surface–peptide binding affinityhas been shown to arise from an increase in both electrostatic andhydrophilic interactions.18

Preparation of samples for MALDI mass analysis was per-formed using four methodologies and using three peptides (brady-kinin, bovine insulin and cyctochromec). In general, owing to thehydrophilic nature of the amine-modified surfaces (water contactangles between 35° and 50°), the sample and matrix solutions con-tacted the entire surface of the polymer disks during evaporation.The resulting crystalline sample consisted of a visually homoge-neous film spread evenly across the polymer disk surface. In allcases MALDI mass spectra were acquired on a laboratory-builtlinear time-of-flight mass spectrometer which has been describedin detail previously.1 Ions were formed using 337 nm radiationand extracted using a continuous 18 kV potential across a sin-gle stage of acceleration. After exiting a¾1 m drift region, ionswere typically detected using a triple microchannel plate detec-tor (except for the case of sample preparation method 2 appliedto bovine insulin, when a dual microchannel plate detector wasemployed). Mass spectra were typically signal averaged on adigital storage oscilloscope for 20 laser shots and subsequentlydownloaded to a personal computer for analysis.

In sample preparation method 1, 2.0µl of analyte solution(0.01 mg ml�1 for bradykinin, 0.30 mg ml�1 for bovine insulin and1.0 mg ml�1 for cytochromec) prepared in aqueous phosphate-buffered saline (PBS) (pH 7.4) was applied to the modified polymerdisks and allowed to dry for¾1 h. Subsequently, 2µl eachof ˛-cyano-4-hydroxycinnamic acid (˛CHCA) (15 mg mL�1) inmethanol and aqueous 10% (v/v) trifluoroacetic acid (TFA) werecodeposited on the probe tips and allowed to dry for an additional¾1 h. This method is analogus to the method used in our previousstudies. For a given surface modification duty cycle, a total of20 MALDI mass spectra were acquired on three separate samples.This process was repeated for each of the three surface modificationduty cycles. Table 1 gives the average integrated protonated analyteion signal observed for each of the three peptides on each of thethree surface modification duty cycles and the standard deviationof these values. In addition, the average percentage decrease inthe protonated analyte ion signal relative to that observed on the3/5 ms modified PET surface is indicated in parentheses.

In sample preparation method 2, 2µl of analyte solution(0.01 mg ml�1 for bradykinin, 0.30 mg ml�1 for insulin and1.0 mg ml�1 for cytochromec) prepared in aqueous PBS and 2µleach of˛CHCA in methanol and aqueous 10% TFA were code-posited on the modified polymer disks and allowed to dry for¾1 h. MALDI mass spectra were acquired using the proceduredescribed above and the average integrated protonated peptide

CCC 1076–5174/99/111205–03 $17.50 Received 23 April 1999Copyright 1999 John Wiley & Sons, Ltd. Accepted 23 August 1999

1206 JMS LETTERS

Table 1. Integrated protonated peptide MALDI ion signals for samples prepared usingmethod 1 on allylamine r.f. pulsed plasma-modified PET surfaces

r.f. pulsed plasma duty cycle (ms)Peptide 3/5 3/15 3/45

Bradykinin 8.0š 2.9 5.0š 1.4 (63%) 3.7š 1.0 (46%)Bovine insulin 14.6š 6.0 11.1š 3.1 (76%) 7.4š 3.0 (51%)Cytochrome c 0.57š 0.25 0.31š 0.15 (54%) 0.20š 0.11 (35%)Average % decrease (64%) (44%)

Table 2. Integrated protonated peptide MALDI ion signals for samples prepared usingmethod 2 on allylamine r.f. pulsed plasma-modified PET surfaces

r.f. pulsed plasma duty cycle (ms)Peptide 3/5 3/15 3/45

Bradykinin 7.6š 2.7 5.1š 2.5 (67%) 3.2š 1.1 (42%)Bovine insulin 5.8š 1.9 4.5š 1.4 (78%) 3.0š 1.3 (52%)Cytochrome c 0.49š 0.12 0.37š 0.12 (76%) 0.30š 0.11 (61%)Average % decrease (74%) (52%)

ion signals determined. Table 2 gives the results of the describedexperiment.

It should be noted that the bovine insulin mass spectra wereacquired using a dual microchannel plate detector, rather thanthe triple microchannel plate detector used for all other MALDIanalyses. Despite the reduced absolute intensities of the bovineinsulin ion signals, the relative trends in the ion signal intensitieswith changes in surface modification duty cycle are consistentwith all other measurements.

In sample preparation method 3, 2µl of analyte solution(0.01 mg ml�1 for bradykinin, 0.30 mg ml�1 for insulin and1.0 mg ml�1 for cytochromec) prepared in methanol and 2µlof ˛CHCA in methanol were codeposited on the modified poly-mer disks and allowed to dry for¾30 min. MALDI mass spectrawere acquired using the procedure described above and the aver-age integrated protonated peptide ion signals determined. Table 3gives the results of the described experiment.

Finally, in sample preparation method 4, 2µl of ˛CHCAin methanol were deposited on the modified polymer disksand allowed to dry. Subsequently, 2µl of analyte solution(0.01 mg ml�1 for bradykinin, 0.30 mg ml�1 for insulin and1.0 mg ml�1 for cytochromec) prepared in methanol were applied

to the matrix coated polymer surface and allowed to dry. MALDImass spectra were acquired using the procedure described aboveand the average integrated protonated peptide ion signals deter-mined. Table 4 gives the results of the described experiment.

From the data presented in Tables 1–4 it is immediately clearthat the peptide MALDI ion signals decrease as the r.f. pulsedplasma duty cycle is changed from 3/5 to 3/15 to 3/45 ms. Consis-tent with our previous studies, these results support the conclusionthat increases in surface–peptide binding affinity, which resultfrom increases in the modified polymer surface amine content,lead to decreases in peptide MALDI ion signals. Interestingly,nearly identical trends are observed for each of the three peptidesstudied and for all four methods of sample preparation. There isperhaps a small enhancement of the influence of surface-peptidebinding when using sample preparation method 1, but the vari-ability of the integrated peptide MALDI ion signals make thisconclusion difficult to confirm.

Alternative approaches designed to block interaction of thepeptide with the substrate surface (similar to sample prepara-tion method 4) could be pursued. However, successful MALDIis generally thought to involve co-crystallization of the matrixand peptide,19,20 thus requiring that both species be dissolved

Table 3. Integrated protonated peptide MALDI ion signals for samples prepared usingmethod 3 on allylamine r.f. pulsed plasma-modified PET surfaces

r.f. pulsed plasma duty cycle (ms)Peptide 3/5 3/15 3/45

Bradykinin 9.9š 4.8 5.5š 2.2 (56%) 5.0š 2.1 (51%)Bovine insulin 12.9š 4.5 7.8š 1.6 (60%) 4.8š 1.5 (37%)Cytochrome c 0.21š 0.08 0.16š 0.05 (76%) 0.15š 0.07 (71%)Average % decrease (64%) (53%)

Table 4. Integrated protonated peptide MALDI ion signals for samples prepared usingmethod 4 on allylamine r.f. pulsed plasma-modified PET surfaces

r.f. pulsed plasma duty cycle (ms)Peptide 3/5 3/15 3/45

Bradykinin 7.2š 1.8 4.8š 1.2 (67%) 4.3š 0.8 (60%)Bovine insulin 13.2š 4.1 10.7š 4.3 (81%) 7.7š 3.3 (58%)Cytochrome c 0.37š 0.14 0.28š 0.10 (76%) 0.15š 0.05 (41%)Average % decrease (75%) (53%)

Copyright 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 1205–1207 (1999)

JMS LETTERS 1207

in a single solvent at some point during sample preparation.This requirement severely restricts the approaches which mightbe used to prevent peptide interaction with the substrate sur-face. More fruitful approaches to overcoming the influence ofsurface–peptide binding might be found in the use of sub-strate surfaces with intrinsically low surface–peptide bindingaffinities and/or the use of chemically aggressive methods forthe dissolution of the surface-bound peptides during samplepreparation.

Cumulatively, the results indicate that the simple changesin solvent and order of matrix/peptide deposition employed inthese studies do not substantially eliminate binding of peptidesto the polymer surfaces and the consequent influence on thepeptide MALDI ion signal. This result is perhaps not surprisinggiven that surface–peptide interactions are known to be extremelyrapid and robust.21 Further, the observation that surface–peptidebinding influences peptide MALDI ion signals under what aregenerally considered to be ‘conventional’ sample preparationconditions (methods 2 and 3) suggests that these effects areroutinely encountered.

The authors thank the NSF for grants BES-9812708 andCHE-9876249 and the Texas Higher Education CoordinatingBoard—Advanced Technology Program for grant 003656-137 forpartial support of this work.

Yours,

CATHY CHEN, ANGELA K. WALKER, YULIANG WU,RICHARD B. TIMMONS and GARY R. KINSEL*Department of Chemistry and Biochemistry, University of TexasArlington, Arlington, Texas 76019-0065, USA

References1. Walker AK, Wu Y, Timmons RB, Nelson KD, Kinsel GR. Anal.

Chem. 1999; 71: 268.

2. Vestling MM, Fenselau C. Mass Spectrom. Rev. 1995; 14:169.

3. Lee SH, Ruckenstein E. J. Colloid Interface Sci. 1988; 125:365.

4. Mizutani T. J. Colloid Interface Sci. 1981; 82: 162.5. Fukuzaki S, Urano H, Nagata K. J. Ferment. Bioeng. 1995;

80: 6.6. Arnebrant T, Nylander T. J. Colloid Interface Sci. 1987; 122:

557.7. Reipa V, Gaigalas A, Abramowitz S. J. Electroanal. Chem.

1993; 348; 413.8. Yu J-L, Anderson R, Ljungh A. J. Surg. Res. 1996; 62: 69.9. Stanislawski L, De Nechaud B, Christel P. J. Biomed. Mater.

Res. 1995; 29: 315.10. Gok E, Kiremitci M, Ates IS. React. Polym. 1994; 24: 41.11. Rabinow BE, Ding YS, Qin C, McHalsky ML, Schneider JH,

Ashline KA, Shelbourn TL, Albrecht RM. J. Biomater. Sci.Polym. Ed. 1994; 6, 91.

12. Hillenkamp F, Karas M, Beavis RC, Chait BT. Anal. Chem.1991; 63: 1193A.

13. Savage CR, Timmons RB, Lin JW. Adv. Chem. Ser. 1993;236: 745.

14. Panchalingam V, Chen X, Savage CR, Timmons RB, Eber-hart RC. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1994; 54:123.

15. Beyer D, Knoll W, Ringsdorf H, Wang J-H, Timmons RB,Sluka P. J. Biomed. Mater. Res. 1997; 31: 181.

16. Rinsch CL, Chen X, Panchalingam V, Savage CR, Wang J-H,Eberhart RC, Timmons RB. Polym. Prepr. 1995; 36: 95.

17. Calderon JG, Harsch A, Gross GW, Timmons RB. J. Biomed.Mater. Res. 1998; 42: 597.

18. Walker AK, Qiu H, Wu Y, Timmons RB, Kinsel GR. Anal.Biochem. 1999; 271: 123.

19. Xiang F, Beavis RC. Rapid Commun. Mass Spectrom. 1994;8: 199.

20. Westman A, Huth-Fehre T, Demirev P, Sundqvist BUR. J.Mass Spectrom. 1995; 30: 206.

21. Magdassi S, Kamyshny A. In Surface Activity of Proteins.Magdassi S (ed). Marcel Dekker: New York, 1996; 1 38.

Copyright 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 1205–1207 (1999)