2JQ4
Anal. Chem. 198% 61, 2504-2511
Coaxial Continuous Flow Fast Atom Bombardment in Conjunction with Tandem Mass Spectrometry for the Analysis of Biomolecules Leesa J. Deterding, M. Arthur Moseley,’ and Kenneth B. Tomer* Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 James W. Jorgenson* Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
The capcl#lty oflnt.rfrolng coawlal contlnwusfkw fast atom bombardment (CF-FAB) wlth tandem mass opectnnnetry (MS/MS) Is drmondratd. The goal of thIs research Is to demonstrate the aMllty of obtaining on-the-fly (Le. chromat o g r a w real t h e ) MS/MS spectra of Momdecules and lo demonstrate the feaslbHlty of uslng open tubular CF-FA6 as a means of Introducing and malntalning a constant flux of a w e into ”amq ” e t e r o v e r long pdodsof tlme. On-the-ttl( MWMS spectra of a tripepH&, Met-Leu-Phe, were obtalned on a 220-pg Injection and a 22-pg Injection. Wnh a total acqukltlon t h e of 2 s, fragment ions resulting from common badrbone cleavages were ObsCHved. Wtth a 50 tun i.d. packed mlorocaplary cokmn,#lo mparatkn d a mbrhm, was obiahred and the MS/MS spectra were acquired as the analytes were eluting from the column. Through the use of the coaxial CF-FAB Interface to ddlver a constant fiow of analyte, MWMS 8pectra of a varlety of COmPOWWIS, lnduding - 9 m f r r t t y ad- - ---
A
-
W
K
-
O-
58
t
_.
. . im
I58
+
2m
I
.,
1
,
~
258
M/Z
Figure 3. MS/MS spectrum (A) of the (M H)+ ion of dipalmRoylphosphatMyIcholine,(8) of the (M H)- ion of palmkoleic acid, (D) of the (M - H)- ion of corticosterone.
times varied from 3 s to 68 min with the total amount of analyte consumed varying from 10 ng to 3.6 wg. It should be noted that this study was not designed to test the limits of detection. Reasonable data can be acquired on significantly lower amounts (i.e., lower by 1-2 orders of magnitude). The MS/MS spectrum of 35 ng of bradykinin acquired over 43 s is representative of the peptide data (Figure 2A). No interpretable MS/MS spectrum could be obtained from this level loaded on the direct insertion FAB probe. As a comparison the MS/MS spectrum of 1hg of bradykinin acquired
r,
.
398
-4
358
+ H)+ ion of maltotetrose, (C) of the (M -
from the direct probe is shown in Figure 2B. These spectra are similar to other published CAD spectra of bradykinin (23). Typical examples of other MS/MS spectra acquired are presented in Figure 3. These data show that the open tubular CF-FAB/MS interface can be used to introduce a sufficiently high flux of analyte over the time period necessary to obtain good-quality MS/MS data. The capability of acquiring MS/MS spectra over long periods of time is demonstrated in Figure 4. The MS/MS spectrum of the (M + H)+ ion of N-acetyl angiotensin I was
ANALYTICAL CHEMISTRY, VOL. 61, NO. 22, NOVEMBER 15, 1989
0
0
II CH,-C-NH
181 95 98 85
2509
0
(hi C=O
NH
OH
&NH
[kH,),
yh OH
CHI
kN
88 75 78 f 65 68 3 55 58 45 F 48
g
z
9
9 35 0: 38
25 28 15
a5 a4
I6
I
5
M/Z Flgurs 4. MS/MS spectrum of the (M -t H)+ ion of N-acetyl angiotensin I acquired over 68 min.
100 w
u z a 0 z 3 m a
1
~
-
: -
50-
318
b2
318'
,
Flgurs 5. MS/MS/MS spectrum of m l r 318 originating from the reaction sequence m l z 466
Table I. Mass Assignment of the Daughter Ions of m / z 217,245, and 279 of Met-Leu-Pheand for the Daughter Ion of m / z 527 of Bradykinin at Varying Scan Speeds
scan rate, sfdecade 100' lo* 5' 3d 2e
-
m l z 318 from Phe-Gly-Leu-Met-NH,.
Table 11. Compounds for Which MS/MS Spectra Have Been Acquired by Using Coaxial Continuous Flow FAB in Conjunction with Tandem Mass Spectrometry
mfz 217
mfz 245
mfz 279
mfz 527
compound
217.0 217.0 217.0 217.4 217.7
244.7 244.8 244.9 245.3 245.7
278.7 278.8 278.9 279.3 279.7
526.6 526.7 527.0 527.5 530.2
Met-Leu-Phe dihydrostreptomycin bradykinin (acidified) maltotetrose corticosterone taurolithocholicacid-3-sulfate
Average of five single scans for m f z 217, 245, and 279; measured from signal averaged data of six scans for m f z 527. * Average of 11 single scans for m f z 217, 245, and 279; measured from signal averaged data of 12 scans for m f z 527. 'Average of 22 single scans for m f z 217, 245, and 279; measured from signal averaged data of 23 scans for m f z 527. dAverageof 25 single scans for m f z 217,245, and 279; measured from signal averaged data of 25 scans for m f z 527. eAverage of 22 single scans for mfz 217, 245, and 279; measured from signal averaged data of 22 scans for m / z 527. a
acquired over 68 min and is representative of 3.6 pg of material. This demonstrates that the coaxial CF-FAB interface is useful in obtaining MS/MS data over long as well as short acquisition time periods.
dipalmitoylphosphatidylcholine
palmitoleic acid N-acetyl angiotensin I
pos ion time,s amt 2 3 43 10 42 45 27
22 pg 33 ng 38 ng 110 ng 231 ng 495 ng 2.3 pg
4070
3.6 fig
neg ion time,s amt 335
1.8pg
246 156 230
2.7pg 1.7~g
2.5 pg
MS/MS/MS Acquisition. In addition to its capability for obtaining MS/MS spectra of biomolecules, the potential of this coaxial CF-FAB interface for the acquisition of MS/MS/MS data has also been studied. We have shown previously that MS" experiments are feasible using the VG ZAB-4F mass spectrometer (11). By following the fragmentation pattern, one can verify the sequence of building blocks within a biomolecule. These experiments, however, can be cumbersome if the daughter ions are of relatively low abun-
2510
ANALYTICAL CHEMISTRY, VOL. 61, NO. 22, NOVEMBER 15, 1989
“1
Met-ENKEPHALIN
li
I ?3
14 48
’2 01
1l
i1
23
’’
eter was scanned at 50 s/decade for a period of approximately 6 min. The analyte concentration was 5 mg/mL,and the flow rate was 50 nL/min. Therefore, approximately 500 ng of material was used for the acquisition of the MS/MS/MS spectrum. MS/MS from Column Separation. Using the packed microcapillary column, a separation of a mixture of Metenkephalin and Met-Leu-Phe was obtained and is shown in Figure 6. The peaks represent 2 pmol of Met-enkephalin and 3 pmol of Met-Leu-Phe, and peak widths at one-half peak height are on the average of 40 s. The MS/MS spectra of the analytw (Figure 7) were obtained while the m888 spectrometer was scanned at 5 s/decade. Fragmentations resulting from typical backbone cleavages were observed for both of the analytes studied.
Met-Leu-Phe
I
3-
14
at
‘5
25
+
‘8 $5
‘IRt
CONCLUSIONS
Figure 6 . Overlald mass chromatograms of the (M H)+ ions of Met-enkephalin (2 pmol) and Met-Leu-Phe (3 pmol) acquked from a
Coaxial continuous flow FAB has proven to be very useful for combining liquid chromatography with tandem mass spectrometry. The ability to obtain on-the-fly MS/MS data makes this coaxial CF-FAB interface compatible with time restraints encountered in chromatography. With the use of packed microcapillary columns, the MS/MS spectra can be obtained on the analytes of a mixture as they are separated and elute from the column. In addition, the MS/MS spectra of a variety of compounds, such as peptides, sugars, fatty acids, phospholipids,and steroids, have been acquired by delivering a constant flow of analyte into the mass spectrometer. This constant flux of analyte can be advantageous for the acquisition of CAD spectra that are otherwise cumbersome (e.g., MS/MS/MS spectra).
separation uslng a packed microcaplllary column.
dance. Much time can be involved due to the need to replenish the FAB probe tip with fresh sample. Therefore, the constant flow of analyte to the probe tip via a continuous flow FAB probe makes this interface ideal for such experiments. The sequencing of the tetrapeptide Phe-Gly-Leu-Met-NH, is shown in Figure 5. Upon collisional activation, the (M H)’ ion of m/z 466 fragments to give the ion of m/z 318, which corresponds to the loss of HMetNH2. In the MS/MS/MS experiment (Figure 5), the (M + H - HMetNH2)+ion fragments to give the loss of CO and the Leu residue (fragment b2). The daughter ion of m / z 120 (fragment al) corresponds to the loss of CO from the Phe residue. The mass spectrom-
+
1Ht
m
U
b.7
I (M+H)+
50
1
2ie
25e
3ie M/Z
358
sire
Figure 7. MSlMS spectra of the (M 4- H)+ Ion of (A) Met-enkephalin (single scan) and (6)Met-Leu-Phe (summation of five scans) acquked from the separation of the mixture uslng the coaxial CF-FAB interface wlth a packed microcapillary column.
Anal. Chem. 1989, 61, 2511-2515
ACKNOWLEDGMENT The packed microcapillary column was made by Ms. Nancy Bragg at the University of North Carolina
