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9+
100
(A)
(B)
8+
10+
5+
Electrospray
Tip
100
6+
7+
To MS
50
50
Electrospray
Solvent
0
Analyte
952
954
m/z
0
(C)
(D)
1000
1500
2000
m/z
1+
100
100
50
50
To MS
0
Matrix
Analyte
2+
8500
8600
m/z
0
5000
10000
m/z
FIGURE 3
Ionization techniques for proteomics. (A) Schematic of ESI. (B) ESI-Orbitrap mass spectrum of bovine ubiquitin.
Inset: high-resolution spectrum of the 9
charge state of bovine ubiquitin illustrating isotopic resolution. (C) Schematic of
MALDI. (D) MALDI-TOF mass spectrum of bovine ubiquitin. Inset: low-resolution spectrum of the 1
þ
þ
charge state of bovine
ubiquitin.
and ease of coupling with LC for online separa-
tions before MS detection.
In ESI, analyte in solution is introduced into
a mass spectrometer via an inlet that sepa-
rates atmosphere from the instrument
molecules in the mass spectrum with a different
mass-to-charge ratio (
m/z
).
33
To promote the gener-
a ionofions,thepHoftheESIsolventis
often adjusted with a small amount (0.1
e
1% v/v)
of acid or base for positive and negative ion
modes, respectively. In positive-ion ESI, the
m/z
is
equal to the molecular mass of the molecule with
the addition of 1 Da for each proton present
divided by
z
. For biomolecules such as proteins,
ESI typically forms a distribution of highly proton-
ated species in the low
m/z
region of a spectrum
(typically an
m/z
of 400
e
3,000), as shown in the
mass spectra for bovine ubiquitin (~8.6 kDa) in
Figure 3
B. This behavior is in contrast to MALDI
spectra, where
z
s vacuum
system. Polypeptide analyte molecules are sus-
pended in an organic/aqueous solution that is
then passed through a small capillary emitter posi-
tioned near themass spectrometer inlet. A 1 to 4 kV
potential difference between the emitter tip and
the inlet creates a
'
ne sprayof small highly charged
droplets that enter the vacuumsystem (
Figure 3
A).
ESIisincontrasttoMALDI,inwhichanalyte
typically starts in a solid phase prior to exposure
to photons that support desorption and gas-
phase ionization (
Figure 3
C). In ESI, the ionized
species form by mechanisms related to solvent
evaporation, droplet
4(
Figure 3
D). High charge
multiplicity has bene
<
ts pertaining to improved
resolution,mass accuracy, andMS/MS fragmenta-
tion patterns (see below); therefore, a variety of
investigations have sought to identify chemical
agents that lead to enhanced analyte protonation
(i.e., super-charging).
34
fission caused by Coulombic
explosion of shrinking chargeddroplets, and evap-
oration of charged molecules directly from the
droplet surface, creating a distribution of charged
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