Letter
pubs.acs.org/ac
High-Sensitivity N‑Glycoproteomic Analysis of Mouse Brain Tissue by
Protein Extraction with a Mild Detergent of N‑Dodecyl β‑D-Maltoside
Jing Liu,†,‡ Fangjun Wang,*,† Jiawei Mao,†,‡ Zhang Zhang,†,‡ Zheyi Liu,†,‡ Guang Huang,†,‡ Kai Cheng,†,‡
and Hanfa Zou*,†
†
Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China
‡
University of Chinese Academy of Sciences, Beijing 100049, China
S Supporting Information
*
ABSTRACT: N-dodecyl β-D-maltoside (DDM), a mild detergent with the ability to maintain
the enzyme activity and solubilize hydrophobic proteins without changing their structures, was
applied for N-glycoproteomic analysis of minute protein sample from mouse brain tissue. After
combining with the capillary-based glycoproteomic reactor, 281 N-glycosylation sites were
successfully characterized from 50 μg of mouse brain tissue, which was 110% higher at least
than those obtained by conventional strategies.
P
buffers with urea and conventional detergents (SDS, Triton X100, and RapiGest SF) for minute protein sample extraction.
Furthermore, as DDM is compatible with trypsin digestion, the
tedious detergent removal steps were avoided and the protein
sample can be online purified by a reversed phase trap column
before liquid chromatography coupled with tandem mass
spectrometry (LC−MS/MS) analysis. Combining this new
protein sample extraction strategy with our previous reported
capillary-based N-glycoproteomic reactor,18 we have characterized 281 N-glycosylation sites corresponding to 170 Nglycoproteins from only 50 μg of mouse brain tissue, which is
about 2 times the numbers identified by using conventional
sample preparation strategies.
We first optimized the concentration of DDM within the
lysis buffer. Mouse brain tissues (about 5−10 mg for each)
were lysed using the lysis buffers with 0, 0.1%, 0.25%, 0.5%, 1%,
and 2% DDM at the ratio of 10 μL buffer/1 mg tissue,
respectively. The protein concentration of each lysate was 5.0 ±
0.2, 6.5 ± 1.0, 7.2 ± 1.0, 7.5 ± 1.1, 9.0 ± 0.5, and 8.8 ± 0.6 mg/
mL, respectively (Supporting Information, Figure S1 and Table
S1). The protein concentration first increased along with the
increase of DDM concentration and reached to a maximum
after the DDM concentration was ≥1%. Therefore, lysis buffer
with 1% DDM was utilized in our following experiments. Then,
we investigated the performance of protein extraction by using
rotein extraction, including the lysis of cells or tissues,
release of proteins from different cell compartments, and
solubilization of proteins into extraction buffer, is the first step
of protein sample preparation.1,2 Usually, the transmembrane
proteins are too hydrophobic to extract into the aqueous
solution, and strong detergents and chaotropes are necessary to
disrupt the membranes and increase the protein extraction
efficiency.3−5 Strong detergents, such as sodium dodecyl sulfate
(SDS),6 are most widely utilized in the extraction of proteins
with high hydrophobicity, such as the glycosylated transmembrane proteins. However, the strong detergents would
significantly decrease the efficiency of enzyme digestion,7,8
chromatography separation, and mass spectrometry detection.
Thus, removing the detergent by protein precipitation with
organic solvents, dialysis, gel filtration, or the recently
developed filter-aided sample preparation (FASP)6 is necessary
before protein digestion and MS detection. Unfortunately, the
process of detergent removal usually induces significant protein
sample loss due to the buffer exchange and redissolving steps,
which compromises the sensitivity, reliability, and accuracy of
proteomic analysis especially for the post translational
modifications (PTMs) analysis of a minute amount of protein
samples.3,9
N-Dodecyl β-D-maltoside (DDM) is a nonionic detergent
composed of a lauryl hydrophobic chain and a maltose
hydrophilic part. It is a mild detergent with the ability to
maintain the enzyme activity,10 purify membrane proteins,11,12
and solubilize hydrophobic proteins without changing their
structures.13−17 Here, we demonstrated the lysis buffer with
DDM and urea exhibited comparable performance to the
© 2015 American Chemical Society
Received: December 18, 2014
Accepted: February 3, 2015
Published: February 3, 2015
2054
DOI: 10.1021/ac504700t
Anal. Chem. 2015, 87, 2054−2057
Letter
Analytical Chemistry
seven different lysis buffers. Briefly, TEAB buffer, TEAB buffer
containing 8 M urea, TEAB buffer containing 8 M urea and 1%
detergent (Triton X-100, RapiGest SF or SDS), TEAB buffer
containing 4% SDS,1,6 and TEAB buffer containing 8 M urea
and 1% DDM were utilized. Finally, the protein concentration
of each lysate was 2.6 ± 0.3 (TEAB buffer), 4.9 ± 0.2 (8 M urea
buffer), 8.6 ± 1.0 (8 M urea buffer with 1% Triton X-100), 9.0
± 1.2 (8 M urea buffer with 1% RapiGest SF), 9.1 ± 0.7 (8 M
urea buffer with 1% SDS), 8.8 ± 0.3 (4% SDS buffer), and 8.9
± 0.4 (8 M urea buffer with 1% DDM) mg/mL, respectively
(Figure 1 and Supporting Information, Table S2). Obviously,
Table 1. Sequence Coverages and Numbers of Unique
Peptides Identified from 1 μg of BSA Digests with Different
Concentration of DDM
DDM concn (mg/mL)
sequence coverage (%)
unique peptides
0
0.1
0.2
0.5
1
2
5
10
77.3
78.3
77.3
78.7
76.4
78.4
74.0
74.6
55
52
51
52
53
51
50
52
the relative standard deviation (RSD) for peptides retention
time were all <2% (Supporting Information, Table S3).
Further, it was observed the DDM was eluted from the LC
column only at a high concentration (80%) of ACN
(Supporting Information, Figure S2). This can be attributed
to the DDM that is retained onto the head of the C18 LC
column during the peptide separation process as the main
peptides separation range is 0−35% ACN in the RP binary
gradient separation. On the other hand, the peak intensity of
DDM was also relatively low even if 10 μg DDM was loaded,
due to it is nonionic detergent and is hard to be ionized. Then,
identical protein samples extracted from HeLa cells were also
digested in the presence of DDM with a different concentration
(Supporting Information, Experimental Section), and similar
results were also obtained among different conditions
(Supporting Information, Table S4, Figures S3 and S4).
Moreover, the good compatibility of DDM with other widely
used enzymes, such as Glu-C, Lys-C (Supporting Information,
Table S5) and PNGase F (Supporting Information, Figure S5),
was also demonstrated. Therefore, the above results demonstrated that DDM has little influence on the enzyme activity,
LC separation, and MS detection and is compatible with
proteomic analysis without any detergent removal steps in
sample preparation procedures.
We also compared the performance of proteomic analysis by
using the DDM strategy as described above with different
protein sample preparation strategies using different detergent
(Triton X-100, RapiGest SF, and SDS) followed with relevant
detergent removing steps (precipitation, acidification, and
FASP) (Supporting Information, Experimental Section).
Finally, it was observed that the sample preparation with
DDM extraction exhibited better performance than those
methods (Precipitation, RapiGest, and FASP) with conventional detergent (Triton X-100, RapiGest SF, and SDS)
extraction followed with relevant detergent removal procedures
(precipitation, acidification, and FASP) in proteomic analysis of
15 μg of mouse brain tissue, and 340 (32%), 126 (10%), and
178 (15%) more proteins were identified (Supporting
Information, Tables S6 and S7).
Protein glycosylation is one of the most important posttranslational modifications and plays a crucial role in a series of
physiological and pathological processes, such as cell−cell
recognition and communication, protein folding, stabilization,
and translocation.19,20 Aberrant N-glycosylation on some
important proteins is highly associated with various types of
cancers.21,22 N-glycosylated proteins are also important for
disease diagnosis, prognosis, and therapeutic response to
drugs.23 Various methods have been developed for in-depth
characterization of the N-glycoproteome, such as hydrazide
Figure 1. Protein extraction efficiency by using lysis buffers containing
different detergents for mouse brain tissues and the protein recovery
after detergent removal. (Precipitation method was used for 1% triton
X-100/8 M urea buffer and 1% SDS/8 M urea buffer; FASP method
was used for 4% SDS buffer).
the protein extraction performance of the buffer with DDM and
urea was comparable to the buffers containing urea and other
commonly used detergents (Triton X-100, RapiGest SF, and
SDS), and all of them were much better than urea buffer or
merely TEAB buffer without detergent. Furthermore, we also
quantitatively investigated the protein loss during the detergent
removal by precipitation and FASP for the protein samples
extracted from 1 mg of tissue. After detergent removal, the
protein concentration of each lysate was 4.8 ± 0.4 (1% Triton
X-100/8 M urea buffer with precipitation), 3.7 ± 0.4 (1% SDS/
8 M urea buffer with precipitation), and 5.1 ± 0.1 (4% SDS
buffer with FASP) mg/mL (Figure 1 and Supporting
Information, Table S2), which demonstrated about 40−60%
protein samples were lost during the detergents removal steps.
Then, we investigated whether DDM is compatible with
trypsin activity and MS detection. The standard BSA proteins
were digested with trypsin and analyzed by LC−MS/MS in the
presence of a different concentration of DDM (Supporting
Information, Experimental Section). After database searching, it
was observed that the numbers of unique peptides identified
and protein sequence coverage were almost not changed along
with the increase of DDM concentration from 0 to 1%
(equivalent to 10 mg/mL) (Table 1). The extracted-ion current
(XIC) of 14 random selected BSA peptides were checked and
marked within the base peak chromatograms with different
DDM concentrations (Figure 2). Obviously, the peak intensity,
peptides elution profiles, and LC separation windows of these
tryptic BSA peptides were all highly similar to each other and
2055
DOI: 10.1021/ac504700t
Anal. Chem. 2015, 87, 2054−2057
Letter
Analytical Chemistry
Figure 2. Base peak chromatograms of LC−MS/MS analysis of 1 μg of BSA digest with the presence of 0 (A), 0.1 (B), 1.0 (C), and 10 (D) mg/mL
DDM. (Peptide peaks labeled by “∗”: (1) HLVDEPQNLIK, (2) KVPQVSTPTLVEVSR, (3) RHPEYAVSVLLR, (4) RPCFSALTPDETYVPK, (5)
KQTALVELLK, (6) SLHTLFGDELCK, (7) LVNELTEFAK, (8) SHCIAEVEKDAIPENLPPLTADFAEDKDVCK, (9) LFTFHADICTLPDTEK,
(10) QTALVELLK, (11) LGEYGFQNALIVR, (12) TVMENFVAFVDK, (13) MPCTEDYLSLILNR, and (14) DAFLGSFLYEYSR).
Table 2. Numbers of Identified N-Glycosylation Sites and N-Glycoproteins by Using Different Protein Sample Preparation
Strategies
experiment 1
experiment 2
total
sample preparation
glyco-proteins
glycosylation sites
glyco-proteins
glycosylation sites
glycosylation sites
glyco-proteins
urea extraction
precipitation method
DDM extraction
72
91
188
55
66
123
111
96
194
78
68
125
134
130
281
95
88
170
chemistry for human plasma and urine,24,25 filter aided sample
preparation for mouse plasma and other organs,23 and so on.
However, usually large amount of staring material is required
for these methods, which limits their application for analysis of
minute amount of samples, such as clinical samples. As most of
the N-glycosylated proteins are hydrophobic transmembrane
proteins, we further investigated if the protein extraction buffer
with DDM can improve the performance of N-glycoproteomic
analysis for minute protein samples from mouse brain tissue.
The reversed phase-hydrophilic (RP-HILIC) capillary-based
glycoproteomic reactor18 was utilized for N-glycoproteomic
sample preparation in this study. Finally, 281 N-glycosylation
sites from 170 glycoproteins were characterized from protein
samples extracted from 50 μg of mouse brain tissue (Table 2
and Supporting Information, Table S8). Comparing with the
sample preparation without any detergent and with strong
detergent followed with precipitation purification, the numbers
of identified N-glycosylation sites and glycoproteins were
increased 110%, 116% and 77%, 93%, respectively (Table 2),
and the sample preparation with DDM extraction covered most
parts of the glycoproteins identified by the other two methods
(69% and 84%, as shown in Figure 3). As the hydrophobic part
of the RapiGest SF will precipitate and block the capillary
column during acidification for detergent removal and part of
the protein will be retained onto the filter membrane and
cannot be loaded onto the glycoproteomic reactors for FASP
strategy, the RapiGest SF and FASP strategies were not
compatible with the RP-HILIC capillary-based glycoproteomic
Figure 3. Venn diagram showing the overlap of the glycoproteins
identified by three different sample preparation strategies. (In the
parentheses were the average Gravy values of proteins in each region).
reactor. After the transmembrane helices prediction (TMHMM
Server v. 2.0, http://www.cbs.dtu.dk/services/TMHMM), 117
glycoproteins (69%) obtained by protein extraction with DDM
were predicted to have at least one transmembrane helices,
which was much better than the other two sample preparation
methods with 53 (56%) and 58 (66%) transmembrane
glycoproteins (Supporting Information, Table S9). Gene
Ontology (GO) analysis was further performed to identify
the cellular location of the identified glycoproteins. For the
DDM strategy, 119 proteins (70%) were membrane proteins,
which was also better than the urea (61 (64%) membrane
proteins) and precipitation (63 (72%) membrane proteins)
strategies, respectively. These results were consistent with the
protein transmembrane analysis and further indicated the better
2056
DOI: 10.1021/ac504700t
Anal. Chem. 2015, 87, 2054−2057
Analytical Chemistry
■
performance of sample preparation method based on DDM on
the analysis of membrane glycoproteins. After calculation of
Gravy value of the glycoproteins identified by different sample
preparation approaches, it was observed the glycoproteins
identified by DDM extraction only were the most hydrophobic
ones with an average Gravy value of −0.21 (Figure 3). This
could be attributed to the low hydrophobic protein extraction
efficiency for protein extraction without any detergent and the
significant sample loss during precipitation and the redissolving
process for protein precipitation. Therefore, the advantage of
DDM in protein extraction for N-glycoproteomic analysis of
minute protein sample is more obvious than in common
proteomic analysis due to most of the glycoproteins are
hydrophobic transmembrane proteins, which are hard to be
redissolved into protein digestion buffer after detergent removal
by protein precipitation.
Protein precipitation or ultrafiltration is unavoidable to
eliminate the interference of detergents on enzyme digestion in
conventional protein sample preparation approaches. However,
sample loss is unavoidable during this detergent removal
procedure, and the purified protein pellet is also hard to fully
redissolve into the aqueous solution without detergent. This
type of protein sample loss greatly compromises the performance of proteomic analysis, especially for the PTMs analyses of
minute protein samples. DDM, a mild detergent, compatible
with trypsin digestion and LC−MS/MS analysis, was
successfully applied in the protein sample extraction in this
study. We observed the protein extraction performance with
1% DDM and 8 M urea was comparable to those with 4% SDS
or 8 M urea with conventional detergents (Triton X-100,
RapiGest SF). After combining with the capillary-based
glycoproteomic reactor, 281 N-glycosylation sites were
successfully characterized from 50 μg of mouse brain tissue,
which was 110% and 116% higher than those methods with
urea extraction and strong detergent extraction followed with
protein precipitation, respectively. Thus, we believe DDM has
great potential in PTMs analysis of minute amount of protein
samples.
■
REFERENCES
(1) Maurer, M.; Mueller, A. C.; Wagner, C.; Huber, M. L.;
Rudashevskaya, E. L.; Wagner, S. N.; Bennett, K. L. J. Proteome Res.
2013, 12, 1040−1048.
(2) Switzar, L.; van Angeren, J.; Pinkse, M.; Kool, J.; Niessen, W. M.
A. Proteomics 2013, 13, 2980−2983.
(3) Ning, Z. B.; Seebun, D.; Hawley, B.; Chiang, C. K.; Figeys, D. J.
Proteome Res. 2013, 12, 1512−1519.
(4) Sun, L. L.; Tao, D. Y.; Han, B.; Ma, J. F.; Zhu, G. J.; Liang, Z.;
Shan, Y. C.; Zhang, L. H.; Zhang, Y. K. Anal. Bioanal. Chem. 2011, 399,
3387−3397.
(5) Chen, E. I.; McClatchy, D.; Park, S. K.; Yates, J. R. Anal. Chem.
2008, 80, 8694−8701.
(6) Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Nat. Meth.
2009, 6, 359−362.
(7) Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S. P.; Gebler, J. C.
Anal. Chem. 2003, 75, 6023−6028.
(8) Chen, E. I.; Cociorva, D.; Norris, J. L.; Yates, J. R. J. Proteome Res.
2007, 6, 2529−2538.
(9) Wang, N.; Xu, M.; Wang, P.; Li, L. Anal. Chem. 2010, 82, 2262−
2271.
(10) Rosevear, P.; Vanaken, T.; Baxter, J.; Fergusonmiller, S.
Biochemistry 1980, 19, 4108−4115.
(11) Loo, T. W.; Clarke, D. M. J. Biol. Chem. 1995, 270, 21449−
21452.
(12) Hazarda, A.; Montemagno, C. Arch. Biochem. Biophys. 2002,
407, 117−124.
(13) Whitelegge, J. P.; le Coutre, J.; Lee, J. C.; Engel, C. K.; Prive, G.
G.; Faull, K. F.; Kaback, H. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
10695−10698.
(14) le Coutre, J.; Whitelegge, J. P.; Gross, A.; Turk, E.; Wright, E.
M.; Kaback, H. R.; Faull, K. F. Biochemistry 2000, 39, 4237−4242.
(15) Morgner, N.; Kleinschroth, T.; Barth, H. D.; Ludwig, B.;
Brutschy, B. J. Am. Soc. Mass Spectrom. 2007, 18, 1429−1438.
(16) Aivaliotis, M.; Karas, M.; Tsiotis, G. J. Proteome Res. 2007, 6,
1048−1058.
(17) Lee, S. C.; Bennett, B. C.; Hong, W. X.; Fu, Y.; Baker, K. A.;
Marcoux, J.; Robinson, C. V.; Ward, A. B.; Halpert, J. R.; Stevens, R.
C.; Stout, C. D.; Yeager, M. J.; Zhang, Q. H. Proc. Natl. Acad. Sci.
U.S.A. 2013, 110, E1203−E1211.
(18) Liu, J.; Wang, F. J.; Lin, H.; Zhu, J.; Bian, Y. Y.; Cheng, K.; Zou,
H. F. Anal. Chem. 2013, 85, 2847−2852.
(19) Helenius, A.; Aebi, M. Science 2001, 291 (5512), 2364−2369.
(20) Lowe, J. B. Cell 2001, 104 (6), 809−812.
(21) Kobata, A.; Amano, J. Immunol. J. Cell Biol. 2005, 83 (4), 429−
439.
(22) Woods, R. J.; Edge, C. J.; Dwek, R. A. Nat. Struct. Biol. 1994, 1
(8), 499−501.
(23) Zielinska, D. F.; Gnad, F.; Wisniewski, J. R.; Mann, M. Cell
2010, 141 (5), 897−907.
(24) Ishihara, T.; Fukuda, I.; Morita, A.; Takinami, Y.; Okamoto, H.;
Nishimura, S. I.; Numata, Y. J. Proteomics 2011, 74 (10), 2159−2168.
(25) Halim, A.; Nilsson, J.; Ruetschi, U.; Hesse, C.; Larson, G. Mol.
Cell. Proteomics 2012, 11 (4), M111.013649.
ASSOCIATED CONTENT
S Supporting Information
*
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
Letter
AUTHOR INFORMATION
Corresponding Authors
*Phone: +86-411-84379576. Fax: +86-411-84379620. E-mail:
[email protected].
*Phone: +86-411-84379610. Fax: +86-411-84379620. E-mail:
[email protected].
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors greatly appreciate Dr. X. Liang and Dr. Z. Guo for
providing the HILIC materials as a gift. Financial support is
gratefully acknowledged for the China State Key Basic Research
Program Grant (Grants 2013CB911203 and 2012CB910601),
the financial support from the Creative Research Group Project
by NSFC (Grant 21321064), and the National Natural Science
Foundation of China (Grants 21235006, 21305139, and
81161120540).
2057
DOI: 10.1021/ac504700t
Anal. Chem. 2015, 87, 2054−2057