TECHNICAL ARTICLE
Accurate Mass Analysis of Intact Ribosomal Proteins
was centrifuged for 15 min at 13,000 rpm and the
supernatant was collected into a sample vial ready for
LCMS analysis.
LCMS conditions
BACKGROUND
Bottom-up proteomic studies rely on the identification of
proteins by the accurate mass and fragmentation analysis
of peptides generated by proteinase digests of isolated
proteins or complex protein mixtures. While this approach
‘identifies’ which proteins are present, the information
about the intact protein(s) is lost. Although SDS-PAGE
provides some information about the protein’s apparent
mass, it is not accurate enough to give an indication of
whether the protein is modified or not.
With the recent advances in high resolution MS instruments,
accurate mass analysis of intact proteins is now routine.
INTRODUCTION
The ribosome is a cluster of proteins responsible for the
biosynthesis of peptides and proteins. It translates the
genetic information in the form of messenger RNA into a
sequence of amino acids. The ribosome has a small and
a large subunit, each containing a number of individual
proteins. The total number of proteins in the 80s ribosome
is ~49 for the large subunit and ~33 for the small
subunit. Recent studies have suggested that ribosomal
proteins maybe involved in functions other than protein
biosynthesis, such as pathological events or developmental
defects. Since ribosomal proteins are relatively small (Mw=
6,000 to 40,000), they can be rapidly identified by accurate
LC MS analysis of the intact proteins.
In the present application ribosomal proteins isolated from
rat liver were separated on a ProteCol™ C8 HQ1003 column.
Sample preparation
80S ribosomal proteins were isolated from a rat liver
microsomal preparation (Williamson et al; 1997,
Eur. J. Biochem. 246: 786-793). One optical density
unit at 260 nm of 80S ribosomal proteins was mixed
with 2 volumes of 6M Guanidine HCl to denature
the proteins. 1 % (v/v) formic acid was subsequently
added to precipitate the nucleic acids. The mixture
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LC: Agilent 1100 LC system
Detection: Agilent 6220 ESI-TOF LC/MS Mass
Column: SGE ProteCol ™ C8 HQ1003, 150 x 2 mm
3 µm 1000 Å (part number 250170)
Flow rate: 0.25 mL/min
Solvent A: Aqueous 0.1 % (v/v) Formic Acid
Solvent B: Acetonitrile / 0.1 % (v/v) Formic Acid
Gradient: 0 min 5 % B
80 min 45 % B
81 min 85 % B
82 min 85 % B
83 min 5 % B
88 min 5 % B
Data Analysis AND RESULTS
All data were acquired and reference mass corrected via a
dual-spray electrospray ionisation (ESI) source. Each scan
or data point on the Total Ion Chromatogram (TIC) is an
average of 15,000 transients, producing a spectrum every
second. Mass spectra were created by averaging the scans
across each peak and background subtracted against the
first 10 seconds of the TIC. Acquisition was performed
using the Agilent Mass Hunter software version B.02.01 and
analysis was performed using Mass Hunter version B.03.01
The resulting base peak chromatogram shows very
high peak capacity - 119 discrete protein masses were
identified; 46 of which were identified as 80S ribosomal
proteins. In some cases several different masses of the
same protein were identified which correlated with known
N- and/or C-terminal processing.
Summary
The ProteCol ™ C8 HQ1003 is the ideal LC column
for intact protein analysis due to its intermediate
hydrophobicity, but most importantly its wide 1000 Å
pore size which enables fast analyte diffusion.
Acknowledgements:
SGE acknowledges Dr Nicholas Williamson and Paul
O’Donnell from Bio21 Mass Spectrometry and Peptide
Synthesis, Bio21 Molecular Science and Biotechnology
Institute, The University of Melbourne, Parkville, Victoria,
Australia for the preparation, chromatography and
analysis of the ribosomal proteins.
TECHNICAL ARTICLE
SGE wishes to acknowledge BIO21 for their collaboration on this application.
No. RT MassProtein
[min]
TA-0141-H © SGE Analytical Science Pty Ltd 09/2010
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
22.4
24.2
26.5
28.7
31.9
36.7
36.7
37.9
42
42.3
42.3
43.8
44.6
45.6
46.6
10943
10943
6648
6276
12321
17279
9399
9270 18449
17623 17779
18448
15667
12122
15644
8087
L37
L37
S30
L39
L36a(L44)
L26
S27a
S27a; cleaved C-term.
L21 NG to KR
L24 & L24 cleaved C-term.
L21
L27
L36
L28
L38
15
16
16
17
18
19
20
21
21
22
23
24
25
26
27
28
29
30
47.3
47.3
47.7
49.5
49.5
50
51
51.6
52.2
53.4
53.9
54.5
55.3
55.3
56.2
56.7
56.7
57.8
12465
23922
23922
27908
14164
18343
14421
15465
15466
15727
28680
23191 23647
23345
16503
24015
9170
14776
15954
L35a
L13 (terminal KK)
L13 (terminal KK)
L8
L31
S11
L35
S24
S24
L32
S6
L14 (native & with mod)
L13a
L27a
L15
S21, N-acetylmethionine
L23 N-acetylserine
S19
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
60
60.8
61.4
63.2
63.2
64.5
64.5
65.8
66.8
67.4
69.5
71.6
72.5
75.3
77
78.5
13284
21527
29464
29862
16314
29466
15379
17091
17629
22169
22460
14708
21893
11772
30355
26585
S20
L18
S4
L7a
S16
S4
S17
S13
S18 N-acetylserine
S7 N-acetylmethionine
S9
S15a
L9
P2
L7
S3 N-acetylalanine
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