Biology Reference
In-Depth Information
Table 1
Total protein and phosphorylation analysis of proteins in the Adenovirus type 2 virion using mass
spectrometry
Sequence
coverage
(%)
c
Number of
phosphorylation
sites detected
d
Number
per virion
a
MS
detection
b
Abbreviation
Protein name
pII
Hexon
720
22
37
3
pIII
Penton base
60
3
17
1
pIIIa
Capsid protein precursor pIIIa
60
14
41
12
pIV
Fiber
36
7
24
1
e
pV
Core protein V/major core
protein
157 ± 1
8
23
2
52K
Encapsidation protein 52K
2
12
2
pVI
Capsid protein precursor pVI
360
5
36
2
pVII
pVII protein
833 ± 19
2
14
2
pVIII
Capsid protein precursor
pVIII
127 ± 3
3
22
3
pIX
Capsid protein IX
240
1
9
1
pTP
DNA terminal protein
2
3
6
1
pX/p
μ
Late L2 mu core protein
125-160
-
-
-
Protease
Protease
~20
5
19
-
a
Combined data from Flint et al. [
1
] and Stewart et al. [
3
]
b
Number of unique tryptic peptides detected in total protein analysis of the Ad2 particle with
p
< 0.05
c
Sequence coverage of proteins after total protein analysis of the Ad2 particle
d
Verifi ed phosphorylation sites, additional phosphorylated sequences might have also been observed [
16
]
e
Only phosphorylated sequence detected, phosphosite not revealed
many PTMs, namely, acetylated [
25
] and ubiquitinated lysines
[
26
], glycosylation (using lectin affi nity columns) [
27
], and tyro-
sine phosphorylation [
23
]. Overall, phosphorylation is one of the
most commonly studied PTMs, and enrichment of phosphoryla-
tion can be obtained in many ways. Apart from antibody-based
affi nity techniques, chemical affi nity is widely used, e.g., immobi-
lized metal affi nity chromatography (IMAC), metal oxide affi nity
chromatography (MOAC), and titanium dioxide as reviewed by
Dunn et al. [
28
]. Moreover, chemical modifi cations with further
surface enrichment have also been developed based on phos-
phoramidate chemistry (PAC) [
29
] and Michael addition [
30
].
Below we describe the titanium dioxide affi nity approach from GL
Sciences Inc., which we have used for identifying phosphorylation
sites in the proteome of Ad2 virions. As reviewed by Thingholm
et al. there are other alternatives than those described here for
loading and elution buffers [
31
].
