This article was downloaded by: [University of Glasgow]On: 18 December 2014, At: 18:53Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

Analytical LettersPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/lanl20

Exact Mass FAB and EI/FAB MS with Peptides Using aDirect Insertion FAB ProbeC. J. Metral a & R. A. Day aa Department of Chemistry , Cincinnati, Ohio, 45211Published online: 05 Dec 2006.

To cite this article: C. J. Metral & R. A. Day (1986) Exact Mass FAB and EI/FAB MS with Peptides Using a Direct Insertion FABProbe, Analytical Letters, 19:1-2, 217-227, DOI: 10.1080/00032718608066253

To link to this article: http://dx.doi.org/10.1080/00032718608066253

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of theContent. Any opinions and views expressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon andshould be independently verified with primary sources of information. Taylor and Francis shall not be liable forany losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use ofthe Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

ANALYTICAL LETTERS, 19(1&2), 217-227 (1986)

EXACT MASS FAB AND EI/FAB MS WITH PEPTIDES USING A DIRECT INSERTION FAB PROBE

C. J. Metral and R. A. Day University of Cincinnati

Department of Chemistry Cincinnati, Ohio 4521 1

ABSTRACT

W e have successfully installed and tes ted the first direct insertion probe

(DIP) fas t a tom bombardment (FAB) gun on a Kratos MS-80 mass spectro-

meter. The installation requires only minor modifications on t h e standard,

EI/CI source which do not interfere with normal EI or CI operation. A small 2

inch extender must be added to t h e solid inlet to accomodate the ex t ra length

of the probe tip; this can be replaced in a few minutes by t h e standard seal to

accept t h e regular solid probe. W e have operated the source under simul-

taneous EI/FAB conditions with excellent results. The use of sorbitol syrup

helps to keep source contamination and background spectrum to a minimum

making it possible t o operate at sufficiently high resolution in order to make

e x a c t mass operation routine. The performance evaluation h a s been carried

out with synthetic peptides and t h e results are presented here.

INTRODUCTION

The most common implementation of FAB in a KRATOS MS-80 mass

spectrometer requires the installation of a FAB gun, removal and replacement

of t h e EI source, and purchase of a sample probe at a relatively high cost. A

more convenient and in many aspects superior a l ternat ive is Phrasor

217

Copyright 0 1986 by Marcel Dekker, Inc. 0003-271 9/86/1901-02 17$3.SO/O

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

218 METRAL AND DAY

1 Scientific's a l l in one DIP GUN . Some of t h e immediate advantages a r e t h e

ease of calibration of the mass scale and unperturbed EI/CI operation. In

addition, the unmodified source makes i t possible t o run CI/FAB and EI/FAB at

high resolution. Using sorbitol syrup as the liquid matrix, i t is possible to

obtain FAB spectra from room temperature up to 100°C with l i t t le source or

spectrum contamination,

EXPERIMENTAL

Instrument Modifications

2

The DIP gun (Phrasor Scientific, Inc.) is shown in FIG. 1. There a r e

basically two modifications necessary to properly mount t h e gun in the Kratos

MS-80 mass spectrometer. First, the orifice in t h e source block tha t allows

the sample into the source cavity must be drilled through to remove a small

taper in the cavi ty side and t h e capillary holder cup removed. Second, a small

removable extender must b e added to t h e solid inlet to accomodate t h e ex t ra

length of the FA6 gun tip. These modifications a r e shown in FIG 2. Phrasor

Scientific can provide a smaller diameter tip which will not require any

modifications of t h e source block but with reduced sensitivity.

DIP GUN Operation

Prior to operating t h e machine under FAB conditions, it is necessary to

cool the source block to 80°C when using sorbitol syrup as the liquid matrix or

to room temperature in the case of glycerol. This c a n b e done without

breaking vacuum, but i t takes too long. A b e t t e r approach is to isolate t h e

source and use the make-up He gas from t h e CC (or nitrogen via t h e reference

inlet) to bring t h e source chamber t o atmosphere. After about 20 minutes, t h e

source should b e a t t h e right temperature. After reestablishing high vacuum,

the machine is calibrated in the normal way under El conditions using

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

Fig. 1 Schematic of FAB direct insertion probe (DIP) (Courtesy of Phrasor Sci.)

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

220 METRAL AND DAY

Extender Counfer Sink Holes Extender Extender

Cap 122"deep Sleeve I D 17'bt bottom I D 3"at top Sleeve

End View t- 1 755'31

0 Counter Sink Holes

122"deep I D J'ot top I D 17'bt bottom

245"

0 7 0 thick 16" 1 9"

Front Bushing

Material : Teflon

Inner Sleeve

Material : Teflon

Fig. 2 Specification for extender.to adapt DIP to t h e Kratos MS-80.

Perfluoro kerosene (PFK) or any suitable reference compound. In order t o g e t

b e t t e r results, i t is important to tune the instrument at t h e highest mass found

with a S/N ra t io of at least 10. One then prepares t h e substrate solution in

sorbitol or glycerol so t h a t t h e viscosity is high enough to maintain t h e

meniscus in a 45' tilt , i f the viscosity is too low t h e sample will clog t h e path

of the fast a t o m beam and no FAB spectra will be possible. One then proceeds

t o apply about 0.5 1 of t h e sample solution to the FAB gun t ip and follow t h e

normal procedure t o introduce a solid sample into t h e source except t h a t t h e

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

MASS FAB AND EI/FAB MS WITH PEPTIDES 221

filament is kept off, and the instrument is lef t in the standby mode until

introduction is completed. At this point, i t is good to have the d a t a system

ready for acquisition and waiting for t h e return key. One then proceeds

according to manual instructions to establish FAB conditions, put the instru-

ment in use mode and proceeds with acquisition. If one desires simultaneous

EI operation, one simply turns the filament current on in the middle of

acquisition. The change in TIC (FIG. 3A, B) is a good indication of when the

machine was under EI/FAB conditions. Operation under high resolution is

similar except t h a t EI/FAB conditions must exist before t h e d a t a system is

allowed to proceed with acquisition.

Calibration

Calibration of t h e mass scale presented some problems above 900 a.m.u.

because of the lack of a suitable volatile calibration compound. Nevertheless,

one can calibrate up to 2400 a.m.u. using PFK in a n indirect manner. This is

possible because the push button control of the mass range in the MS-80

(acceleration voltage), gives exac t factors of 1.5, 2, 3, and 4 in t h e mass.

Therefore, one simply calibrates up to 600 a.m.u. in the normal mode and use

t h e push button to access t h e higher mass range. The masses read must la ter

b e corrected by using the appropriate factor.

RESULTS

Comparison of Glycerol vs. Sorbitol as a Liquid Matrix

The utility of glycerol as a liquid matrix for many compounds in FAB

mass spectrometry is well established. However, glycerol has some drawbacks

which a r e more pronounced in EI/FAB or high resolution exact mass work.

Major problems a r e related t o i ts low molecular weight combined with a

tendency to form adducts as well as i t s relatively high vapor pressure. The

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

222 METRAL AND DAY

80-

c 70-

5 60- v C 50- 0

40- 0 2 30-

20-

10-

0

c

f!

- - c

001 0 0 7 020 0 3 2 0 4 5 0 5 7 1 1 0 1 2 3 135rnin 100 I I l l l I l . l l

90-

FAB Only m 4 EVFAB- 9 l ' l ' l ' l ' l ' l ' l ' l

0 4 2 0 5 4 1 2 4 1 5 4 2 23 min

90 - 80 -

c 70- C g 60-

a 50- L

I I I 1 0 10 15 20 25

Scan

Fig. 3 Tota l ion cu r ren t (TIC) for (A) t h e pept ide TRP-SER-PHE as function of operational mode. Up to FAB only a f t e r EI/FAB. (8) FAB only and EI/FAB TIC and fragment ions of GLY-ASP.

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

MASS FAB AND EIIFAB MS WITH PEPTIDES 2 2 3

formation of glycerol adducts introduces a large number of mass peaks t h a t

frequently dominate the mass spectrum and complicate interpretation a t

obscure peaks of analytes. The higher vapor pressure results in high source

contamination, rapid loss of sample signal, and in the case of EI/FAB,

suppression of t h e FAB contribution to t h e spectrum as well as t h e reference

compound spectrum. This is especially t rue when operating in the exac t mass

mode. A particularly annoying problem is t h e slow deposition of glycerol a t

the base of the filament which results in a slow shorting of t h e power supply

driving the electron energy. Sorbitol syrup, on t h e other hand, can operate up

to 8OoC without any of these drawbacks producing a much cleaner spectrum.

FAB Spectra of Selected Peptides

The FAB spectra of several available synthetic peptides were taken to

test t h e performance of the DIP gun. Two spectra of the peptide GPGG a r e

shown in FIG. 4; 4A is in glycerol and 4B in sorbitol. The prominent matrix

ions for glycerol a r e fragments m/z 57, 75; MH' at 93; and cluster ions a t 185

and 277; for sorbitol m/z 129, 165 and 183. MH' - n H20 a r e t h e only

significant ions from sorbitol. The quasi-molecular ion (m/z = 287) is

prominent in both glycerol (A) and sorbitol (B), but more intense in the latter.

The base peak in t h e glycerol is t h a t of t h e glycerol quasi-molecular ion (m/z =

93) but in sorbitol, i t is t h e proline fragment ion (m/z = 70). The N-terminal

sequence ions, the A series, at m/z 127, 155, 212, and 241; internal f ragments

m/z 114 (GLY-GLY) and 154 (PRO-GLY); and C-terminal fragments, t h e 2

series, m/z 74 and 102 a r e relatively enhanced in sorbitol. The sensitivity of

the machine has not been accurately measured, but we have been able to

acquire usable FAB spectra and is of five nanograms of GPGG in glycerol and

2 or 3 ng for GPGG in sorbitol syrup at 80°C.

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

224 METRAL AND DAY

100

90

80

7 0

60

50

40

30

20

10

0

100

90

80

70

60

50

40

30

20

10

0

93

5 7 7 5

7 0

I,L ' I I I

1

I

'0 100 l !

185

A

2 0 7

1

5

207 B

D 200 250 300 350 400 450

Fig. 4 Mass spectra of GPGC. (A) In glycerol at room temperature . (B) In sorbitol a t room temperature.

The capability of operating a non-interfering matrix at higher temper-

a tures is shown with the spectra of t h e peptide MCMM in glycerol at room T

(Fig. In t h e former, t h e two most 5A) and is sorbitol at 8OoC (Fig. 5B).

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

MASS FAB AND EI/FAB MS WITH PEPTIDES

185 93

80

7 0

60

50

40

30

20

10

0

4

150

L ,.&

100 200

189 100

904 ';o I 80

60

300 400

20 1

3 50

40

30

20

10

0

225

A

a

7 500

B 469

150 200 250 300

0

L 350 400 450 500

Fig. 5 Mass spectra of MGM. (A) In glycerol at room temperature. (8) In sorbital at 80°C.

intense peaks are the quasi-molecular ions of glycerol (GH' m/z 93) and the

dimer (G2H+ m/z 185). In sorbitol, the base peak is the MC, an A' fragment

m/z 189. The other peptide related ions MH' m/z 469; the A+ fragment,

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

226 METRAL AND DAY

Table I. Exact Mass Data for MH' m/z 287 and A f ragment (GPG + H+) m/z

230.

C C13 H N 0 DEV MEAS. MASS BPTS. %INT.

I 1 0 19 4 5 -0.1 287.1354 17 62.60"

9 0 16 3 4 1.4 230.1155 12 31.63*

MGM' m/z 320; 2 t 2 f ragments (GMM + 2H)' at m/z 338, and (MM + 2H)' at

281 a r e relatively intense with weaker sorbitol peaks SH+ m/z 183 and (S - H20

+ H)+ m/z 165. In general, the S/N values in t h e spectral region of interest are

be t te r than with glycerol.

Exact Mass Determination

Exact mass measurements may b e effected in FABMS, many instruments

lack the capability. The DIP GUN source on the MS-80 allows simultaneous EI

and FAB and thus t h e simultaneous acquisition of PFK data for calibration and

FAB spectra of t h e analyte. In Table I a r e partial da ta from this mode of

operation with the peptide GPGG.

Enhancement of Intensities During EI/FAB

Neutrals a r e ionized, e.g. PFK, whether they a r e introduced by another

mode, or a r e produced by FAB. In the la t te r category or have MgIZ releasing

low levels of 12+' but larger amounts of I2 by FAB. The I2 ionizes in EI giving

intense peaks (data not shown). Neutrals related t o ions a r e apparently ionized

by the EI and strongly augment the existing ions (Fig. 3B). There ions m/z 133,

145, 175 increase sharply when the electron beam is turned on.

Discussion and Conclusion

W e have found the EI/FAB operation to offer benefits not readily

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14

MASS FAB AND EI/FAB MS WITH PEPTIDES 22 7

implemented with t h e standard source; one such is the ease of calibration. The

capability of acquiring PFK fragment da ta simultaneously with the acquisition

of the FAB spectra of the analyte allows the implementation of t h e exac t

mass calculation routine. EI/FAB can b e used to analyze volatile species being

formed; for instance, we have found that Mg12 contributes little ion current as

I2 by FAB, but by EI/FAB m/z 254 becomes the base peak. Further studies

must be made t o quant i ta te the sensitivity of the FAB gun with several

substrates in addition t o peptides. An enhancement in sensitivity at the high

mass can be readily accomplished with a post acceleration detection in-

creasing t h e FAB gun usefulness.

The use of sorbitol syrup as a liquid matrix has complemented the

EI/FAB capabilities by removing many interfering peaks and keeping source

contamination low. In addition, sorbitol syrup offers t h e possibility of carrying

out chemical reactions in the FAB probe because of the wider temperature

range of application, an option we a r e exploring.

REFERENCES

1.

2.

J. F. Mahoney, J. Perrel, and Taylor (1984) Am. Lab. 5 92-103.

K. Jayasimhulu, R. A. Day, C. J. Metral, J. Wermeling, and M. E. Madis (1985) Proc. ASMS m, 510-511.

Received November 1 2 , 1985 Accepted November 13, 1985

Dow

nloa

ded

by [

Uni

vers

ity o

f G

lasg

ow]

at 1

8:53

18

Dec

embe

r 20

14