Biology Reference
In-Depth Information
gurations have
been used for ESI.
33,35
Conventional ESI utilizes
solvent
Various experimental con
(the ability to differentiate two adjacent species
within a spectrum), peak capacity (maximum
number of observable species with an analytical
platform), detection limits (the ability to detect
low amounts of analyte), dynamic range (the
ability to simultaneously detect analyte over
a broad concentration range), mass accuracy (the
ability to detect analyte at the correct mass), exper-
imental duty cycle, types of MS/MS techniques,
and cost. To overcome limitations associated
with individual mass analyzers and improve
sampling capacity in proteomics work
L/min with
a counter gas to facilitate desolvation. For pro-
teomics, more sensitive micro-ESI and nano-ESI
methods with lower
flow rates of 1 to 10
m
ow rates (200
e
1,000
nL/min and 10
e
200 nL/min, respectively)
provide performance improvements without
a counter gas
ow.
36,37
Micro- and nano-ESI
generate smaller droplets, which promote desol-
vation and allow the emitter to be placed closer
to the inlet to improve analyte transfer ef
ciency.
ESI is a concentration sensitive technique; thus,
analyte signal does not typically increase with
higher
ows,
modern mass spectrometers are often com-
bined into hybrid instruments (e.g., Q-TOF,
QqQ, QqQ-FTMS, QIT-FTMS) that exploit distinct
advantages of each mass analyzer in tandem.
43
As stated earlier, proteins typically form
a number of highly charged species using ESI.
This approach is advantageous because most
mass analyzers exhibit better resolving power in
the low
m/z
range and many ion detectors
are less sensitive at higher
m/z
. High-resolution
instruments
44
(e.g., FT-ICR and Orbitrap) can
readily resolve molecular ion carbon-12/
carbon-13 isotopic multiplets, as demonstrated in
the spectrum for the
z
ow rates.
33
However, at very low
ow
rates (
100 nL/min), MS signal becomes limited
by the number of gas phase ions produced.
38
ESI-MS can be performed of
<
ine by infusing
the sample directly into the mass spectrometer
with robotic and micro
uidic devices.
39
Alterna-
tively, online MS analysis can be performed as
proteins elute off a high-performance liquid
chromatography (HPLC) column, as discussed
later in this chapter.
40
Micro- and nano-ESI typi-
cally provide detection of proteins with solution
concentrations from 1 to 10
5
ine
ESI-MS. When coupled online with capillary
LC/MS, low attomole levels of intact proteins
can be routinely achieved.
41
nM for of
9 protonated species of
bovine ubiquitin (
Figure 3
B, inset). High resolving
power allows simple assignment of the mole-
cular ion charge state by counting the number
of isotopes in a single
m/z
unit or the
m/z
difference
between adjacent isotopes (
z
¼
m/z
iso1 e iso2
).
Protein molecular mass is often reported as
the monoisotopic (
12
C
100%
13
C
0%
) or most abun-
dant isotopic (
12
C
100-n%
13
C
n%
) mass; these values
are differentiated by an italic number that
follows the reported molecular mass (
Figure 3
B,
inset). This approach is in contrast with
lower-resolution approaches that report average
molecular mass (
Figure 3
D, inset).
2
On a well-
calibrated instrument, individual isotopes can
be assigned with sub part-per-million mass accu-
racy, which greatly improves selectivity of protein
identi
¼
1/
D
Mass Analyzers and the Impact of
Resolution on Intact Protein Analysis
The core of a mass spectrometer is the mass
analyzer, which converts a measureable quantity
(e.g., time, frequency, current) into a plot of
m/z
versus intensity. Today
sstate-o -the-artmass
spectrometers can be divided into four primary
classes:
'
(1) quadrupole (Q);
(2) quadrupole
ion-trap (QIT);
ight (TOF); and
(4) Fourier transform MS (FTMS), which inc-
ludes ion cyclotron resonance (FT-ICR) and
Orbitrap
(3)
time-of-
.
2,42
e
46
The various mass analyzers
used for protein analysis provide distinct
advantages in terms of mass resolving power
cation from theoretical databases, as dis-
cussed later in this chapter. To date, the largest
protein tobe characterizedwith isotopic resolution
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