Biology Reference
In-Depth Information
Table 1
Points to be considered when choosing label-free spectral counting approach
Label-free spectral counting
References
Strong points
Under controlled conditions, spectral count is highly reproducible
between technical replicates
[
11
]
Faster and cheaper when compared to labeling methods
[
1
,
16
-
18
]
No limits for replicates; ideal for low amounts of samples
[
3
]
Straightforward method
[
19
]
Weak points
Inferior accuracy when compared to labeling techniques, especially due
to suppression effect
[
1
]
Data normalization is still challenging
[
18
]
Low discrimination for peptides shared by multiple proteins
[
20
]
Low-abundance proteins might not be detected, especially due to
co-eluted peptides' effect
[
1
,
13
,
21
,
22
,
29
]
label-free experiments is prefractionation, prior to tryptic digestion,
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE). After electrophoresis, gels are stained and the lanes excised
into several smaller segments (the number dependent upon the orig-
inal amount of protein loaded). Each of these segments is then in-
gel digested with trypsin and tryptic peptides contained in each
digested segment are analyzed by reversed-phase LC-MS/MS
(Fig.
1
). The approach is known as GeLC-MS/MS, and it has been
extensively used for label-free spectral counting approaches.
Besides prefractionation, an MS-based strategy extensively
used to detect low-abundance peptides, or co-eluted peptides, is
dynamic exclusion. Dynamic exclusion refers to a particular period
of time where the mass spectrometer ignores the
n
most abundant
ions, previously fragmented, and will select the next most abun-
dant ions for fragmentation. So, if the mass spectrometer is set to
isolate and fragment the three most abundant ions, it will select the
4th, 5th, and 6th most abundant ions for subsequent fragmenta-
tion in the following survey scan [
2
,
9
,
24
]. Zhang and coworkers
mention that 90 s is an optimal value and that the optimal dynamic
exclusion time is proportional to the average chromatographic
peak width at the base of the eluting peptides [
9
]. An alternative
method to overcome the co-elution issue is to change the mass
spectrometer operational mode from data-dependent (DDA) to
data-independent analyses (DIA). This approach was originally
suggested by Venable and Dong [
25
], and does not rely on parent
mass scanning but on a continuous MS/MS scan acquisition in
narrow
m
/
z
ranges until a larger
m
/
z
window is covered.
