Sequence-specific Fragmentation from Oligopeptides Using Plasma-desorption Mass Spectrometry Alexander M. Buko* and Virender K. Sarin Department of Analytical Research, Abbott Laboratories, Abbott Park, IL 60064, USA SPONSOR REFEREE: Professor R. G. Cooks, Purdue University, West Lafayette, IN 47907, USA
='Cf plasma-desorption mass spectrometry (PDMS) has been demonstrated to provide sequence-specific fragmentation for several oligopeptides. The nature of the fragment ions observed is generally similar to that observed using liquid secondary-ion mass spectrometry (LSIMS) and can be observed using less sample than LSJMS requires, but PDMS spectra are acquired at a lower resolution. In addition, the molecular weight of some of the oligopeptides studied exceeds that which is generally accepted as within the sequence range of LSIMS. The specific series of sequence ions that predominate in the PDMS spectra appear to be related to the amino acid compositions and sequences of the oligopeptides.
Recent developments in mass spectrometry have extended the range of bio-organic compounds suitable for mass spectral analysis. The ionization of nonvolatile and thermally labile compounds by plasma-desorption,' liquid secondary-ion,' ion-spray3 and laser-desorption mass spectrometry4, has been demonstrated for molecules of molecular weight exceeding 20 000 Da. In addition to molecular weight information, structural data can also be generated. Sequence analysis of oligopeptides with molecular weights below 2500 Da can be generally accomplished at the nanomole level using liquid secondary-ion mass spectometry (LSIMS)6 and at the sub-nanomole level using LSIMS in combination with collisionally activated dissociation (CAD).',' Generating sequence information from oligopeptides with molecular weights greater than 2500Da has been more difficult. This study demonstrates that sequence information can be observed routinely from oligopeptides with molecular weights between 1300 and 5500 Da using 252Cfplasmadesorption mass spectrometry. EXPERIMENTAL Muss spectra. LSIMS mass spectra were obtained using an MS-50 mass spectrometer (Kratos, Manchester, UK) equipped with an ion gun made by Ion-Tech (Teddington, UK). Xenon was used for the ion beam and the spectra were recorded at 6 kV acceleration potential. A 1:l (v/v) thioglycerol+ glycerol mixture was used as the sample matrix. The plasma-desorption (PD) mass spectra were obtained using a BIOION 20 time-of-flight mass spectrometer (Bio-Ion Nordic AB, Uppsala, Sweden). PD mass spectra were accumulated over a time equivalent of 5 x lo6 start counts (typically 30 min) using an acceleration voltage of 19 kV and a flight tube length of 14 cm. Masses calculated for molecular ions are isotopically averaged. From 0.5 to 1.0 milligrams of sample was dissolved in an appropriate spreading solvent (n-propanol + 40% formic acid (l:l, v/v) or ethanol+50% 0.1 M trifluoroacetic acid (l:l, v/v)) to yield solutions of 2 nmol per 10 pL. * Author to whom correspondence should be addressed.
~
Table 1. Amino acid sequences of oligopeptides studied Peptide
Bapp-1 60262 Glucagon Big gastrin I H33F
Amino acid sequence
DAEFRHDSGYEVHHQKLVFFAEDVGSNK EQNIRDSFQKVTLRRY RK HSQGTFTSDYSKYLDSRRAQDFVQWLMNT < ELGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDF-NH2
KKPDYEPPVVHGCPLPPPKSPPVPPPRKKRTW LTESTLSTALAELATRSF
Mellitin Angiotensin I Leu-paracelsin" Bovine insulin Renin substrate SPB-5378 Pal-SPBb SPC-134
GIIAVLKVL'TTGLPALISWIKRKRQQ-NH2 DRVYIHPFHL Ac-BABABBQBLBGBBPVBBQQ-J
GIVEQCCASVCSLYQLENYCN/FVNQHLCGSHLVEALYLVCGERGFFYTPKA DRVYIHPFHLLVYS YSVILLDTLLGRMLPQLVCRLVLRCS Pal-YSVILLDTLLGRMLPQLVCRLVLRCS
GIPCCPVHLKRLLIVVVVVVLIVVVIVGALL-
MGL Pal-GIPCCPVHLKRLLIVVWVLIVVVIVGALLMGL a B = 2-methylalanine (Aib); J = phenylalaninol (Pheol). Pal = Dalmatic acid.
Pal-SPCb
Subsequently, 10 pL of peptide solution was deposited onto a nitrocellulose target. After drying, the target was rinsed once with distilled water or 0.1% trifluoroacetic acid. Peptideslproteins. A 51-amino-acid peptide, H33F, and two small hydrophobic pulmonary surfactant peptides SPB-5378 and SPC-134 along with the corresponding palmitic-acid-modified peptides Pal-SPB and Pal-SPC were synthesized at Abbott Laboratories. Two small hydrophilic peptides BAPP-1 and 60262 were also synthesized at Abbott Laboratories. H33F, SPB-5378, SPC-134, Pal-SPB, Pal-SPC, BAPP-1 and 60262 are names given by Abbott Laboratories for specific sequences (see Table 1). Glucagon, big gastrin I and mellitin were obtained from Peninsula Labs Inc. (Belmont, CA, USA). Bovine insulin, renin substrate decapeptide and angiotensin I were obtained from Sigma Chemical Co. (St Louis, MO, USA). Leu-12 paracelsin was a gift from Dr R. Chen of Abbott Laboratories.
0951-4198/90/0541-0545$05.oO
0 John Wiley & Sons Limited, 1990
RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 4, NO. 12, 1990 541
SEQUENCE-SPECIFIC FRAGMENTATION FROM OLIGOPEPTIDES
fragment ions that are observed in this study include A,, B, and Y: ions. The A, and B, ions are N-terminal fragment ions (Fig. l(a) and l(b)), whereas, the Y:ions are C-terminal fragment ions (Fig. l(c)). A typical LSIMS mass spectrum may contain several sequencespecific ions of these types using 0.5 to over 1nmol of peptide. Figure 2(a) displays the LSIMS mass spectrum from 2 nmol renin decapeptide. Several A,-type fragment ions are observed. Using CAD in conjunction with a triple-stage quadrupole or 4-sector mass spectrometer can increase the amount of sequence information and may decrease the amount of sample for analysis from 500 down to less than 50 pmol.*," However, the use of CAD in conjunction with tandem mass spectrometers for generating sequence specific ions is still generally limited to oligopeptides of molecular weight below 2500Da.' It would be useful to be able to do sequence studies on larger peptides without resorting to the usual multiple hydrolysis to provide overlapping smaller peptides. Molecular ions of oligopeptides can often be observed by PDMS using 10 to 100 pmol of sample for
(C)
+
513
0
514
H,N - C - C - N - C - COOH H H H
Figurel. Nomenclature for sequence ions in mass spectra of
peptides.'
RESULTS AND DISCUSSION The types of sequence ions obtained from oligopeptides using mass spectrometry have been described in detail by Roepstorff.' Fragment ions can include the C-terminus or N-terminus of a peptide (Fig. 1). Those
50001
(C)
4000(
3wM 2
P Y
E
P
20wc
10000
L 0
mlz 0
m
imoa
B
I Y
12000
M
80;L
40W
mlz
,
€00
800
1000
rioo
Figure2. Spectra from LSIMS of (a) 2 nmol renin substrate; (b) 50 pmol renin substrate; and PDMS of (c) 2 nmol renin substrate, and (d) 50 pmol renin substrate.
542 RAPID COMMUNICATIONS IN MASS SPECTROMETRY. VOL. 4. NO. 12, 1990
@ John Wiley & Sons Limited, 1990
SEQUENCE-SPECIFIC FRAGMENTATION FROM OLIGOPEPTIDES
Table 2. Analysis of oligopeptides studied BBa
Peptide
Ib
AAC
MWd
Sequence ions‘
Bapp-I 2688 25000 28 3263 A,, n = 5-22 60262 1530 12500 18 2338 Y;:, n=2-13 A,, n=7-10 0 SO00 29 3484 A,, n = 6-25 Glucagon Big gastrin I -1530 5000 34 3851 A,, n = 5-21 H33F -3660 5000 51 5532 Y:, n=3-26 Mellitin -3800 8000 20 2849 Y:, n=3-20 Angiotensin I A,, n = 3-10 -4290 28000 10 1297 Leu-paracelsin -6180 9000 20 1935 B,, n = 1-6,s-10,12,13 Bovine insulin -6620 20000 51 5734 Renin substrate -7700 40000 14 1760 A,,, n=3-13 SPB-5378 -12844 5000 26 2976 Y:, n=3-25 A,,, n=12-26 Pal-SPB -14499 2800 27 3215 Yl, n=3-26 A,, n=10-27 SPC-134 -21012 1800 34 3549 A,, n = 8-30 Pal-SPC A,, n = 9-32 . -22645 2000 35 3788 Bull and Breese hydrophobicity index.’* bIntensity of molecular ion over a time equivalent of 5 x 10‘ start counts. Number of amino acid residues. Calculated isotopically averaged molecular ion [M + HI+ Nomenclature of Roepstorff.’
='Cf plasma-desorption mass spectrometry (PDMS) has been demonstrated to provide sequence-specific fragmentation for several oligopeptides. The nature of the fragment ions observed is generally similar to that observed using liquid secondary-ion mass spectrometry (LSIMS) and can be observed using less sample than LSJMS requires, but PDMS spectra are acquired at a lower resolution. In addition, the molecular weight of some of the oligopeptides studied exceeds that which is generally accepted as within the sequence range of LSIMS. The specific series of sequence ions that predominate in the PDMS spectra appear to be related to the amino acid compositions and sequences of the oligopeptides.
Recent developments in mass spectrometry have extended the range of bio-organic compounds suitable for mass spectral analysis. The ionization of nonvolatile and thermally labile compounds by plasma-desorption,' liquid secondary-ion,' ion-spray3 and laser-desorption mass spectrometry4, has been demonstrated for molecules of molecular weight exceeding 20 000 Da. In addition to molecular weight information, structural data can also be generated. Sequence analysis of oligopeptides with molecular weights below 2500 Da can be generally accomplished at the nanomole level using liquid secondary-ion mass spectometry (LSIMS)6 and at the sub-nanomole level using LSIMS in combination with collisionally activated dissociation (CAD).',' Generating sequence information from oligopeptides with molecular weights greater than 2500Da has been more difficult. This study demonstrates that sequence information can be observed routinely from oligopeptides with molecular weights between 1300 and 5500 Da using 252Cfplasmadesorption mass spectrometry. EXPERIMENTAL Muss spectra. LSIMS mass spectra were obtained using an MS-50 mass spectrometer (Kratos, Manchester, UK) equipped with an ion gun made by Ion-Tech (Teddington, UK). Xenon was used for the ion beam and the spectra were recorded at 6 kV acceleration potential. A 1:l (v/v) thioglycerol+ glycerol mixture was used as the sample matrix. The plasma-desorption (PD) mass spectra were obtained using a BIOION 20 time-of-flight mass spectrometer (Bio-Ion Nordic AB, Uppsala, Sweden). PD mass spectra were accumulated over a time equivalent of 5 x lo6 start counts (typically 30 min) using an acceleration voltage of 19 kV and a flight tube length of 14 cm. Masses calculated for molecular ions are isotopically averaged. From 0.5 to 1.0 milligrams of sample was dissolved in an appropriate spreading solvent (n-propanol + 40% formic acid (l:l, v/v) or ethanol+50% 0.1 M trifluoroacetic acid (l:l, v/v)) to yield solutions of 2 nmol per 10 pL. * Author to whom correspondence should be addressed.
~
Table 1. Amino acid sequences of oligopeptides studied Peptide
Bapp-1 60262 Glucagon Big gastrin I H33F
Amino acid sequence
DAEFRHDSGYEVHHQKLVFFAEDVGSNK EQNIRDSFQKVTLRRY RK HSQGTFTSDYSKYLDSRRAQDFVQWLMNT < ELGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDF-NH2
KKPDYEPPVVHGCPLPPPKSPPVPPPRKKRTW LTESTLSTALAELATRSF
Mellitin Angiotensin I Leu-paracelsin" Bovine insulin Renin substrate SPB-5378 Pal-SPBb SPC-134
GIIAVLKVL'TTGLPALISWIKRKRQQ-NH2 DRVYIHPFHL Ac-BABABBQBLBGBBPVBBQQ-J
GIVEQCCASVCSLYQLENYCN/FVNQHLCGSHLVEALYLVCGERGFFYTPKA DRVYIHPFHLLVYS YSVILLDTLLGRMLPQLVCRLVLRCS Pal-YSVILLDTLLGRMLPQLVCRLVLRCS
GIPCCPVHLKRLLIVVVVVVLIVVVIVGALL-
MGL Pal-GIPCCPVHLKRLLIVVWVLIVVVIVGALLMGL a B = 2-methylalanine (Aib); J = phenylalaninol (Pheol). Pal = Dalmatic acid.
Pal-SPCb
Subsequently, 10 pL of peptide solution was deposited onto a nitrocellulose target. After drying, the target was rinsed once with distilled water or 0.1% trifluoroacetic acid. Peptideslproteins. A 51-amino-acid peptide, H33F, and two small hydrophobic pulmonary surfactant peptides SPB-5378 and SPC-134 along with the corresponding palmitic-acid-modified peptides Pal-SPB and Pal-SPC were synthesized at Abbott Laboratories. Two small hydrophilic peptides BAPP-1 and 60262 were also synthesized at Abbott Laboratories. H33F, SPB-5378, SPC-134, Pal-SPB, Pal-SPC, BAPP-1 and 60262 are names given by Abbott Laboratories for specific sequences (see Table 1). Glucagon, big gastrin I and mellitin were obtained from Peninsula Labs Inc. (Belmont, CA, USA). Bovine insulin, renin substrate decapeptide and angiotensin I were obtained from Sigma Chemical Co. (St Louis, MO, USA). Leu-12 paracelsin was a gift from Dr R. Chen of Abbott Laboratories.
0951-4198/90/0541-0545$05.oO
0 John Wiley & Sons Limited, 1990
RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 4, NO. 12, 1990 541
SEQUENCE-SPECIFIC FRAGMENTATION FROM OLIGOPEPTIDES
fragment ions that are observed in this study include A,, B, and Y: ions. The A, and B, ions are N-terminal fragment ions (Fig. l(a) and l(b)), whereas, the Y:ions are C-terminal fragment ions (Fig. l(c)). A typical LSIMS mass spectrum may contain several sequencespecific ions of these types using 0.5 to over 1nmol of peptide. Figure 2(a) displays the LSIMS mass spectrum from 2 nmol renin decapeptide. Several A,-type fragment ions are observed. Using CAD in conjunction with a triple-stage quadrupole or 4-sector mass spectrometer can increase the amount of sequence information and may decrease the amount of sample for analysis from 500 down to less than 50 pmol.*," However, the use of CAD in conjunction with tandem mass spectrometers for generating sequence specific ions is still generally limited to oligopeptides of molecular weight below 2500Da.' It would be useful to be able to do sequence studies on larger peptides without resorting to the usual multiple hydrolysis to provide overlapping smaller peptides. Molecular ions of oligopeptides can often be observed by PDMS using 10 to 100 pmol of sample for
(C)
+
513
0
514
H,N - C - C - N - C - COOH H H H
Figurel. Nomenclature for sequence ions in mass spectra of
peptides.'
RESULTS AND DISCUSSION The types of sequence ions obtained from oligopeptides using mass spectrometry have been described in detail by Roepstorff.' Fragment ions can include the C-terminus or N-terminus of a peptide (Fig. 1). Those
50001
(C)
4000(
3wM 2
P Y
E
P
20wc
10000
L 0
mlz 0
m
imoa
B
I Y
12000
M
80;L
40W
mlz
,
€00
800
1000
rioo
Figure2. Spectra from LSIMS of (a) 2 nmol renin substrate; (b) 50 pmol renin substrate; and PDMS of (c) 2 nmol renin substrate, and (d) 50 pmol renin substrate.
542 RAPID COMMUNICATIONS IN MASS SPECTROMETRY. VOL. 4. NO. 12, 1990
@ John Wiley & Sons Limited, 1990
SEQUENCE-SPECIFIC FRAGMENTATION FROM OLIGOPEPTIDES
Table 2. Analysis of oligopeptides studied BBa
Peptide
Ib
AAC
MWd
Sequence ions‘
Bapp-I 2688 25000 28 3263 A,, n = 5-22 60262 1530 12500 18 2338 Y;:, n=2-13 A,, n=7-10 0 SO00 29 3484 A,, n = 6-25 Glucagon Big gastrin I -1530 5000 34 3851 A,, n = 5-21 H33F -3660 5000 51 5532 Y:, n=3-26 Mellitin -3800 8000 20 2849 Y:, n=3-20 Angiotensin I A,, n = 3-10 -4290 28000 10 1297 Leu-paracelsin -6180 9000 20 1935 B,, n = 1-6,s-10,12,13 Bovine insulin -6620 20000 51 5734 Renin substrate -7700 40000 14 1760 A,,, n=3-13 SPB-5378 -12844 5000 26 2976 Y:, n=3-25 A,,, n=12-26 Pal-SPB -14499 2800 27 3215 Yl, n=3-26 A,, n=10-27 SPC-134 -21012 1800 34 3549 A,, n = 8-30 Pal-SPC A,, n = 9-32 . -22645 2000 35 3788 Bull and Breese hydrophobicity index.’* bIntensity of molecular ion over a time equivalent of 5 x 10‘ start counts. Number of amino acid residues. Calculated isotopically averaged molecular ion [M + HI+ Nomenclature of Roepstorff.’
