Sequencing of peptides by mass spectrometry

Details Sequencing of peptides by mass spectrometry Sequencing of peptides by mass spectrometry

2001-03-13

2004-09-28

Zeman, Mary K. (Department: 1631)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Biological or biochemical

C702S027000, C530S300000, C530S350000, C250S281000, C250S282000, C250S283000, C250S286000, C250S287000

Reexamination Certificate

active

06799121

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to methods of protein analysis and more particularly concerns methods of sequencing oligopeptides.
BACKGROUND OF THE INVENTION
Identifying the sequence of amino acid residues in a protein is often the first step in a study of the function of an unknown protein. This is traditionally accomplished by means of Edman degradation (Edman (1970)) Despite being a time-honored method, Edman sequencing has a major disadvantage in that it requires, typically, milligram quantities of a protein, an amount often considered excessive in today's laboratory practice and standards. Gas-phase microsequencing by means of tandem mass spectrometry has increasingly been applied to obtain partial, or even complete, sequences of unknown peptides (Hunt et al. (1986); Johnson et al. (1988); Papayannopoulos (1995)). In the last couple of years, a major application of the above has been in the area of proteome analysis, where partial sequences of enzymatically cleaved peptides have been used for the purpose of protein identification via the so-called “sequence tag” method (Shevchenko et al. (1996); Figeys et al. (1996)). Mass spectrometry's major advantages in chemical analysis have been well-documented (McLafferty (1981)) and are applicable to peptide sequencing.
A protonated peptide, produced by means of electrospray (Fenn et al. (1989), fragments typically under low-energy (≦100 eV) tandem mass spectrometric conditions to yield product ions that are indicative of the amino acid residue sequence (Hunt et al. (1986); Johnson et al. (1988); Papayannopoulos (1995)). In solution, the most basic sites on a peptide are the nitrogen atoms at the N-terminal amino group and the side chains of the histidine, lysine, and arginine residues (Cantor et al. (1980); Smith et al. (1991)). However, once the protonated peptide ion has been desorbed into the gas phase, transfer of the “external” proton, particularly after collisional activation, to amidic functional groups, the carbonyl oxygen and the amidic nitrogen atoms, on the backbone becomes possible (McCormack et al. (1993); Jones et al. (1994); Dongré et al. (
Mass Spectrom.
1996); Dongré et al. (
J. Am. Chem. Soc.
1996). The peptide then fragments at the protonated peptide linkage to yield an ionic and a neutral fragment. If the charge is retained on the N-terminal fragment, a b ion is produced; the b ion may subsequently lose CO to form the a ion. Alternatively, if the charge is retained on the C-terminal fragment, a y″ (y+2H) ion is produced (Hunt et al. (1986); Johnson et al. (1988); Papayannopoulos (1995); McCormack et al. (1993); Jones et al. (1994); Dongré et al. (
Mass Spectrom.
1996); Dongré et al. (
J. Am. Chem. Soc.
1996). Results from a series of studies have shown that the b ion has an oxazolone structure (Yalcin et al. (1995); Yalcon et al. (1996); Ambihapathy et al. (1997); Nold et al. (1997)), the a ion is an immonium ion, and the y″ ion is a protonated peptide or amino acid (Hunt et al. (1986); Johnson et al. (1988); Papayannopoulos (1995); McCormack et al. (1993); Jones et al. (1994); Dongré et al. (
Mass Spectrom.
1996); Dongré et al. (
J. Am. Chem. Soc.
1996). A product-ion spectrum of a protonated peptide typically consists of series of b, a, and y″ ions which reflect the amino acid sequence. Unfortunately, the fragmentation chemistry of a peptide is rich, and there may be gaps in the series of product ions; this makes sequencing nonroutine to all but the highly experienced.
The fragmentation of alkali- and alkaline-metal containing peptides in tandem mass spectrometry has been a subject of much interest and study (Renner et al. (1988); Grese et al. (1989); Grese et al. (1990); Hu et al. (1992); Hu et al. (
J. Am Soc. Mass Spectrom.
1993); Hu et al. (
J. Am. Chem. Soc.
1993); Tang et al. (1988); Teesch et al. (1990); Teesch et al. (
J. Am. Chem. Soc.
113, 812-820 (1991)); Teesch et al.
J. Am. Chem. Soc.,
113, 3668-3675 (1991); Zhao et al. (1993); Leary et al. (1989); Leary et al. (1990); Lee et al. (1998)). Although most of these reports concentrated on examination of the structures of metal-containing product ions and the mechanisms of their formation (Renner et al. (1988); Grese et al. (1989); Grese et al. (1990); Hu et al. (1992); Hu et al. (
J. Am Soc. Mass Spectrom.
1993); Hu et al. (
J. Am. Chem. Soc.
1993); Tang et al. (1988); Teesch et al. (1990); Teesch et al. (
J. Am. Chem. Soc.
113, 812-820 (1991)); Teesch et al. (
J. Am. Chem. Soc.,
113, 3668-3675 (1991); Zhao et al. (1993); Leary et al. (1989); Leary et al. (1990); Lee et al. (1998)), the potential for acquiring sequence information from metal-containing, e.g., lithiated and sodiated, peptides was readily apparent irrespective of whether that was implied or explicitly stated (Leary et al. (1989)). Unlike protonated peptides, lithiated and sodiated peptides tend to fragment after collisional activation to yield primarily the [b
n
+OH+X]
+
ions (X=Li or Na) (Renner et al. (1988); Grese et al. (1989); Grese et al. (1990); Tang et al. (1988); Teesch et al. (
J. Am. Chem. Soc.,
113, 3668-3675 (1991); Lee et al. (1998)). These product ions are believed to form from elimination of C-terminal residues from precursor ion structures in which the alkali-metal ion is bound to the C-terminal carboxylate anion of a zwitterionic peptide. Rearrangement as a result of collisional activation results in transfer of the C-terminal OX group to the carbonyl carbon of the preceding residue and elimination of CO and an imine (Renner et al. (1988); Grese et al. (1989); Grese et al. (1990); Lee et ala. (1998)). Unfortunately, the affinities of peptides for the lithium and the sodium ions are weak relative to those for the proton (Bouchonnet et al. (1992); Klassen et al. (1996)); consequently, cleavage of the metal ion is often a more favorable fragmentation pathway relative to backbone fragmentation and leads to low yields in these more sequence-informative reactions.
SUMMARY OF THE INVENTION
The present inventors have found that they are able to determine the sequences of peptides or proteins by analysing the peptides or proteins in argentinated form using mass spectrometry.
Accordingly the present invention provides a method of analyzing argentinated peptides or proteins using mass spectrometry comprising:
(a) combining an oligopeptide with silver to provide a sample comprising argentiated oligopeptide;
(b) submitting the sample to a mass spectrometer;
(c) performing scans of silver containing peaks in optimum collision energies;
(d) identifying any doublet or triplet peak pattern;
(e) confirming with Y ions;
(f) determining partial sequence by the mass separation between two successive doublet or triplet patterns. Preferably the oligopeptide comprises from about 3 to about 10 amino acids and the performing scans comprise collecting product ion spectra of the [M+Ag]
+
ion, where M=oligopeptide;
According to a preferred embodiment, the method of the invention utilizes silver nitrate.
According to another embodiment of the method of the invention according the determination of partial sequence comprises searching for, and identifying cleaved amino acid residues based on differences in m/z values of neighboring triplets where the m/z value of the [b
n
−H+Ag]
+
ion and the corresponding [y
n
+H+Ag]
+
ion are related by the formula: [y
n
+H+Ag]
+
=[M+Ag]
+
+Ag
+
−[b
n
−H+Ag]
+
. Preferably the searching and identifying is conducted by a custom search algorithm, more preferably the algorithm is written in Visual Basic and looks for the triplet peak pattern of (m/z)
1
, (m/z)
2
)=(m/z)
1
−18.0, and (m/z)
3
=(m/z)
2
−28.0 as well as the doublet pattern of (m/z)
2
and (m/z)3, all to within ±0.5 m/z unit.
According to yet another embodiment of the method the product ion spectra of

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