1. Analysis of proteoforms in membrane protein complexes by
top-down proteomics
Contact information
Hans JCT Wessels, PhD
Radboudumc
Nijmegen
The Netherlands
E: hans.wessels@radboudumc.nl
T: +31-(0)24-3616933
Introduction
• Protein complexes are key players in
important cellular processes
• The exact combinations of amino acid
sequence and post-translational modifications
of subunits (proteoforms) in complexes are
mostly unknown
• Native gel electrophoresis can be used to
isolate protein complexes
• Top-Down proteomics enables the
unambiguous characterization of proteoforms
in complex samples
• At present, no method is available to extract
intact proteins from native electrophoresis
gels for Top-Down proteomics
Objective
Development of a robust extraction method for
sensitive characterization of subunit proteoforms
in membrane protein complexes isolated by
native gel electrophoresis.
Results
• Unbiased extraction of subunits
• Top-Down proteomics detected 28 out of 30
subunits (93% coverage)
• Missing subunits were extremely hydrophobic
proteins
• 120 proteoforms could be identified
• Various PTMs were detected such as
phosphorylation, acetylation and formylation
Conclusions
We have established an unbiased extraction
method for Top-Down proteomics of protein
complexes isolated by native gel electrophoresis.
Application of nanoflow LC-UHR Qq ToF MS/MS
enabled sensitive detection of different
proteoforms for nearly all subunits of CIV and CV
from Bovine heart mitochondria.
J. Wessels1, S. Guerrero-Castillo1, R. Tans1, P. Schmit2, K. Marx3, S. Pengelley3, A. van Gool1
¹Radboudumc, Nijmegen (NL); ²Bruker Daltonique S.A., Wissembourg (FR); ³Bruker Daltonik GmbH, Bremen (GER)
Protein complex Subunits Top-Down
subunits
Top-Down
proteoforms
Bottom-Up
subunits
Complex IV 14 12 (86%) 53 14 (100%)
Complex V 16 16 (100%) 67 14 (88%)
Methods
Mitochondrial membrane complexes were
isolated from Bovine heart and separated by
native gel electrophoresis. Gel slices containing
the complexes were cut from the gel to extract
intact subunits. Extracted proteins from each
complex were analyzed by SDS-PAGE to assess
the extraction efficiency. Complexes IV and V
were analyzed by Top-Down and tryptic Bottom-
Up proteomics.
8700 8750 8800 8850 8900 8950 m/z
0
1
2
3
4
5
6
6x10
Intens.
0
1
2
3
4x10
Intens.
8715 8720
ATP5J [AA 33-108]
Compound 15
Retention time: 14.0 min
Measured Mr: 8952.5043Da
Calculated Mr: 8952.4960 Da
Mass error: 0.0083 Da (0.93 ppm)
ATP5J [AA 34-108]
Compound 14
Retention time: 13.9 min
Measured Mr: 8838.4633Da
Calculated Mr: 8838.4531 Da
Mass error: 0.0102 Da (1.16 ppm)
ATP5J [AA 35-108]
Compound 16
Retention time: 14.3 min
Measured Mr: 8710.3599 Da
Calculated Mr: 8710.3581 Da
Mass error: 0.0012 Da (0.21 ppm)
-Asn (N)-Lys (K)
26
21
329
3
5
6
23
30
11
18
14
24
19
7 12 2917 25104 13 2815 22 27 3320 31
1 1682
15
10
2417
32297
4121
12
13
31
28
11
18
26 345 25 4316141 489 39362319 37 4530 3327 448 4022 35 4220 382 3 4 6
0
1
2
3
4
5
6x10
Intens.
0.0
0.5
1.0
1.5
2.0
2.5
7x10
10 12 14 16 18 20 22 Time [min]
Cytochrome c oxidase (CIV)
ATP Synthase (CV)
Extraction method: 100 µg of Bovine heart mitochondrial protein was
separated by native gel electrophoresis. Complexes of interest were cut
from the gel, snap-frozen, crushed and incubated in detergent buffer.
Subsequently, the detergent buffer was exchanged for Urea.
SDS-PAGE: Protein complex extracts were separated by SDS-PAGE and
visualized by silver staining to assess the extraction efficiency at the
subunit-level.
LC-MS/MS: Complex IV and V extracts were analyzed directly by liquid
chromatography with online tandem mass spectrometry (maXis 4G ETD and
Impact II; Bruker Daltonics). 10µl sample (20% of extracted volume) was
loaded onto the trapping column (Acclaim PepMap C4; Thermo Scientific).
Proteins were separated on a 0.150 x 150mm PLRP-S column (Michrom) at
30° C using a 30 minutes linear gradient of 10-80% acetonitrile / 0.1%
formic acid. Data-dependent CID MS/MS spectra were recorded. For
Bottom-Up proteomics complex IV and V extracts were subjected to in-
solution tryptic digestion and analyzed by liquid chromatography with
online tandem mass spectrometry (maXis 4G ETD and amaZon speed ETD;
Bruker Daltonics). Peptides were separated using a 0.075 x 150mm C18 RP
column (Acclaim RSLC 120 C18; Thermo scientific) at 40° C and a 240
minutes linear gradient of 5-45% acetonitrile / 0.1% formic acid. Data-
dependent CID and ETD fragmentation spectra were recorded. Data were
analyzed in DataAnalysis 4.2, BioTools 3.2, ProteinScape 3.1 (Bruker
Daltonics) and MASCOT V2.4 (Matrix Science).
• Multiple subunits showed ragged N-termini
• The equivalent of 10µg of total mitochondrial
protein on column was required to detect the
dominant proteoforms
Figure 2: Extracted ion chromatograms of intact subunits (Dissect features)
of complex IV and V.
Figure 3: (A) Deconvoluted MS spectrum of ATP5J proteoforms with
differentially processed N-terminus. This spectrum shows the charge
deconvoluted (MaxEnt) spectral peaks of the complex V ATP5J subunit with
mitochondrial import sequence cleavage at amino acid positions 33, 34 and
35 of the canonical protein sequence. (B) Annotated charge deconvoluted
(SNAP2) CID MS/MS spectrum in BioTools of the Mr 8952.5043 Da ATP5J
[AA 33-108] subunit.
Figure 1: (A) Coomassie stained native electrophoresis gel of Bovine heart
mitochondrial complexes and (B) silver stained SDS-PAGE of the protein
extracts. The DDM lane of the SDS-PAGE serves as control of each complex
for unbiased extraction. The urea gel lane is the protein extract used for LC-
MSn analysis.
27
21
3
5
6
23
30
11
18
14
24
19
7 2912 17 25
104 2813 15 2622 3320 31
1 16 3282 9
24
327
21
12
13
15
31
28
11
1810
26 345 432516141 489 3917 36
2319 37 4530 3327 448
29 4022
35 424120 382 3 64
0
1
2
3
4
5
6x10
Intens.
0.0
0.5
1.0
1.5
2.0
2.5
7x10
Intens.
10 12 14 16 18 20 22 Time [min]
Cytochrome c oxidase (CIV)
ATP Synthase (CV)
Table 1: Top-Down and Bottom-Up proteomics results
(A) (B) (A)
(B)