Mass Spectometry of Proteins and Peptides [Methods In Molec, Książki, Biochemia
[ Pobierz całość w formacie PDF ]
Methods in Molecular Biology
TM
VOLUME 146
Spectrometry
of Proteins
and Peptides
Edited by
John R. Chapman
HUMANA PRESS
Methods in Molecular Biology
TM
Mass
Mass
Spectrometry
of Proteins
and Peptides
Edited by
John R. Chapman
HUMANA PRESS
Peptide Sequencing by Tandem MS
1
1
De Novo Peptide Sequencing
by Nanoelectrospray Tandem Mass
Spectrometry Using Triple Quadrupole
and Quadrupole/Time-of-Flight Instruments
Andrej Shevchenko, Igor Chernushevich,
Matthias Wilm, and Matthias Mann
1. Introduction
Recent developments in technology and instrumentation have made mass
spectrometry the method of choice for the identification of gel-separated pro-
teins using rapidly growing sequence databases
(1)
. Proteins with a full-length
sequence present in a database can be identified with high certainty and high
throughput using the accurate masses obtained by matrix-assisted laser desorp-
tion/ionization (MALDI) mass spectrometry peptide mapping
(2)
. Simple pro-
tein mixtures can also be deciphered by MALDI peptide mapping
(3)
and the
entire identification process, starting from in-gel
digestion
(4)
and finishing
with acquisition of mass spectra and database search, can be automated
(5)
.
Only 1–3% of a total digest are consumed for MALDI analysis even if the
protein of interest is present on a gel in a subpicomole amount. If no conclusive
identification is achieved by MALDI peptide mapping, the remaining protein
digest can be analyzed by nanoelectrospray tandem mass spectrometry (Nano
ESI-MS/MS)
(6)
. Nano ESI-MS/MS produces data that allow highly specific
database searches so that proteins that are only partially present in a database,
or relevant clones in an EST database, can be identified
(7)
. It is important to
point out that there is no need to determine the complete sequence of peptides
in order to search a database—a short sequence stretch consisting of three to
four amino acid residues provides enough search specificity when combined
with the mass of the intact peptide and the masses of corresponding fragment
From: Methods in Molecular Biology, vol. 146:
Protein and Peptide Analysis: New Mass Spectrometric Applications
Edited by: J. R. Chapman © Humana Press Inc., Totowa, NJ
1
2
Shevchenko et al.
ions in a peptide sequence tag
(8)
(
see
Subheading 3.4.
). Furthermore, pro-
teins not present in a database that are, however, strongly homologous to a
known protein can be identified by an error-tolerant search
(9)
.
Despite the success of ongoing genomic sequencing projects, the demand
for
de novo
peptide sequencing has not been eliminated. Long and accurate
peptide sequences are required for protein identification by homology search
and for the cloning of new genes. Degenerate oligonucleotide probes are
designed on the basis of peptide sequences obtained in this way, and subse-
quently used in polymerase chain reaction-based cloning strategies.
The presence of a continuous series of mass spectrometric fragment ions
containing the C terminus (y
′′
ions. However, it is necessary to obtain addi-
tional evidence that the particular fragment ion does indeed belong to the y
′′
′′
series. To this end, a separate portion of the unseparated digest is esterified
using 2
M
HCl in anhydrous methanol (
Fig. 1A
) (
see
Subheading 3.2.
). Upon
esterification, a methyl group is attached to the C-terminal carboxyl group of
each peptide, as well as to the carboxyl group in the side chain of aspartic and
glutamic acid residues. Therefore the
m/z
value of each peptide ion is shifted
by 14(
n
+ 1)/z, where
n
is the number of aspartic and glutamic acid residues in
the peptide, and
z
is the charge of the peptide ion. The derivatized digest is then
also analyzed by Nano ESI-MS/MS, and, for each peptide, fragment ion spec-
tra acquired from underivatized and derivatized forms are matched. An accu-
rate peptide sequence is determined by software-assisted comparison of these
two fragment spectra by considering precise mass differences between the
adjacent y
ions as well as characteristic mass shifts induced by esterification
(
see
Subheading 3.4.1.
) (
Fig. 2
). Since esterification with methanol signifi-
cantly shifts the masses of y
′′
ions (by 14, 28, 42, ... mass units), it is possible
to use low-resolution settings when sequencing is performed on a triple qua-
drupole mass spectrometer, thus attaining high sensitivity on the instrument.
This sequencing approach employing esterification is laborious and time con-
suming and requires much expertise in the interpretation of tandem mass spec-
′′
Fig. 1. Chemical derivatization for mass spectrometric
de novo
sequencing of pep-
tides recovered from digests of gel separated proteins.
(A)
A protein is digested in-gel
(
see
Subheading 3.1.
) with trypsin and a portion of the unseparated digest is esterified
by 2
M
HCl in anhydrous methanol (
see
Subheading 3.2.
).
(B)
A protein is digested
in-gel
with trypsin in a buffer containing 50% (v/v) H
2
18
O and 50% (v/v) H
2
16
O (
see
Subheading 3.1.
).
(C)
A protein is digested in-gel
with trypsin, and the digest is
esterified and subsequently treated with trypsin in the buffer containing 50% (v/v)
ions)
(10)
has been successfully used to deter-
mine
de novo
sequences using fragment ion spectra of peptides from a tryptic
digest
(11)
. The peptide sequence can be deduced by considering precise mass
differences between adjacent y
Peptide Sequencing by Tandem MS
3
H
2
18
O and 50% (v/v) H
2
16
O (
see
Note 22
). Here, R
1
repesents the side chain of argin-
ine or lysine amino acid residues (these are trypsin cleavage sites) whereas R
x
repre-
sents the side chain of any other amino acid residue except for proline.
4
Shevchenko et al.
Fig. 2. Peptide
de novo
sequencing by comparison of tandem mass spectra acquired
from intact and esterified peptide. A 120-kDa protein from
E. aediculatis
was purified
by one-dimensional gel electrophoresis
(24)
and
digested in-gel
with trypsin; a part of
the digest was analyzed by Nano ESI-MS/MS on an API III triple quadrupole mass
spectrometer (PE Sciex, Ontario, Canada). A separate part of the digest was esterified
and then also analyzed by Nano ESI-MS/MS.
(A)
Tandem (fragment-ion) mass spec-
trum recorded from the doubly charged ion with
m/z
666.0 observed in the conven-
tional (Q1) spectrum of the original digest.
(B)
Matching tandem spectrum acquired
from the ion with
m/z
673.0 (
∆
mass = [673–666]
×
′′
ions in spectrum
B
are shifted by 14 mass units com-
pared with the corresponding y
′′
ions in spectrum
A
.
tra. However, it allows the determination of accurate peptide sequences even
from protein spots that can only be visualized by staining with silver
(12
,
13)
.
An alternative approach to
de novo
sequencing became feasible after a novel
type of mass spectrometer—a hybrid quadrupole/time-of-flight instrument (Q/TOF
[14]
or QqTOF
[15]
)
was introduced. QqTOF instruments allow the acquisi-
tion of tandem mass spectra with very high mass resolution (>8000 full-width
at half-maximum height [FWHM]) without compromising sensitivity. These
instruments also benefit from the use of a nonscanning TOF analyzer that
2 = 14) in the conventional (Q1)
spectrum of the esterified digest. The peptide sequence was determined by software-
assisted comparison of spectra
A
and
B
. The only methyl group was attached to the
C-terminal carboxyl of the peptide (designated by a filled circle) and therefore the
masses of the singly charged y
[ Pobierz całość w formacie PDF ]