Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
pubs.acs.org/ac
Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian 116023, China
DOI: 10.1021/acs.analchem.6b00910
Anal. Chem. 2016, 88, 49714978
Article
Analytical Chemistry
EXPERIMENTAL SECTION
Cell Culture, Metabolic Labeling, Cells and Media
Pretreatment. The MHCC97H, MHCC97L (HCC cells with
high and low metastatic potentials, respectively, kindly
presented by professor Yinkun Liu, Fudan University), and
HeLa cells (ATCC) were grown in a humidied atmosphere of
5% CO2 at 37 C in DMEM media (Thermo) supplemented
with 10% (v/v) FBS (Gibco) and 1% penicillin/streptomycin
(Thermo), respectively. For the medium labeling media (Lys4, Arg-6), L-lysine- and L-arginine-depleted SILAC DMEM
media (Thermo) were supplemented with [4,4,5,5-D4] L-lysine
(100 g mL1, Thermo), [13C6] L-arginine (100 g mL1,
Thermo), L-proline (200 g mL1, Thermo), 10% dialyzed FBS
(Gibco), and a 1% penicillin/streptomycin mixture. For the
heavy labeling media (Lys-8, Arg-10), only [4,4,5,5-D4] Llysine and [13C6] L-arginine were replaced with [13C6, 15N2] Llysine (Thermo) and [13C6, 15N4] L-arginine (Thermo). The
MHCC97L cells were grown in the medium media, and the
MHCC97H cells were grown in the heavy media. In addition,
HeLa cells were labeled with another heavy labeling medium
(Lys-6, Arg-10), which used [13C6] L-lysine and [13C6, 15N4] Larginine.
The SILAC-labeled cells were grown to at least six doubling
times to ensure the complete incorporation of the labeled
amino acids. Passaging was performed when 8090%
conuency was reached. To prepare the CMs from the HeLa
cells, the cells were incubated in complete labeling medium for
24 h. For the MHCC97H and MHCC97L cells, the cells and
CMs were both harvested after 48 h and were mixed based on
the number of cells.
The collected cells were suspended in 6 M guanidine
hydrochloride (Sigma) supplemented with 1% (v/v) protease
inhibitor cocktail (Sigma). Then, cell suspension was ultrasonicated on ice for 200 s in total (10 s intervals every 10 s),
followed by centrifugation at 20 000 rpm at 4 C for 30 min.
The supernatants were collected, and the protein concentration
was determined by a BCA assay (Beyotime, China).
The collected CMs were centrifuged at 500g and 3000g for
15 min to remove cells and cell debris, respectively. After
ltrating through a 0.22 m lter unit (Millipore, MA), the
supernatant was concentrated and desalted with water via
Amicon 3 kDa lter devices (Millipore, MA), increasing the
4972
DOI: 10.1021/acs.analchem.6b00910
Anal. Chem. 2016, 88, 49714978
Article
Analytical Chemistry
(Thermo, MA). All of the samples were stored at 80 C for
further analysis.
For the peptides from MHCC97L and MHCC97H cells,
they were injected onto an Agilent 2100 HPLC system
(Agilent, CA) with a high pH-stable RP column (4.6 mm 250
mm, 5 m, 100 , Durashell, China) at a ow rate of 0.5 mL
min1. The peptides were eluted with a gradient from 5% to
45% solvent B over 55 min (solvent A: 20 mM ammonium
acetate, pH 10; solvent B: acetonitrile, 20 mM ammonium
acetate, pH 10). In all, 50 fractions were collected every 1 min
from 5 to 55 min. Then, fractions with equal collection time
intervals (5 min) were pooled. In this way, ve pooled fractions
were obtained pending further liquid chromatography coupled
with tandem mass spectrometry (LC-MS/MS) analysis.
LC-MS/MS Analysis. The peptides were analyzed with a 1D
nano-RPLC-MS/MS on a Q-Exactive MS (Thermo Fisher
Scientic, CA) coupled with an Ultimate 3000 (Dionex,
Germany) nano-LC system. The mobile phases were buers
A (2% acetonitrile, 98% water, and 0.1% formic acid) and B
(98% acetonitrile, 2% water, and 0.1% formic acid). Fused-silica
capillaries (150 m i.d. 375 m o.d.) were obtained from
Sino Sumtech (Handan, China). A C18 trap column (150 m
i.d. 5 cm) was connected to a homemade capillary separation
column (75 m i.d. 15 cm). Both the trap and separation
columns were packed with Daiso C18 particles (5 m, 100 ;
Osaka, Japan). To separate the peptides from the HeLa CMs, a
short gradient (52 min) was established: 37 min of 6%25%
buer B and then 15 min of 25%35% buer B with a ow rate
of 300 nL min1. To quantify additional proteins from the
MHCC97H and MHCC97L cells and CMs, a 110 min gradient
was established, comprised of 90 min of 6%22% buer B, and
then 20 min of 22%35% buer B. The spray voltage was 2.5
kV, and the temperature of the ion transfer capillary was set at
275 C. The Q-Exactive MS was operated in positive ion data
dependent mode, and the 10 most intense ions were subjected
to HCD fragmentation with normalized collision energy at
28%. The MS1 scans were performed at a resolution of 70 000
from m/z 300 to 1800 (automatic gain control (AGC) value,
1E6; maximum injection time, 100 ms), and the data were
acquired in prole mode. The MS/MS scans were performed at
a resolution of 17 500 (AGC, 1E5; maximum injection time, 60
ms), and the data were acquired in centroid mode using a 20 s
exclusion window. The unassigned ions or those with a charge
of +1 and >+7 were rejected. One microscan was acquired for
each MS and MS/MS scan. A lock mass correction was also
appended using a background ion (m/z 445.12003).
Database Searching. The raw data were uploaded into
Proteome Discoverer (PD, version 1.4.1.14) with Mascot
(2.3.2) and were searched against the UniProtKB human
complete proteome sequence database (release 2015_04,
42,121 entries). The reverse sequences were appended for an
FDR evaluation. The mass tolerances were set at 7 ppm for the
parent ions and at 20 ppm for the fragments. The peptides were
searched using tryptic cleavage constraints, and a maximum of
two missed cleavages were allowed. The minimal peptide length
was six amino acids. Carbamidomethylation (C) (+57.0215
Da) was used as the xed modication. Oxidation (M;
+15.9949 Da) and acetylation (protein N-termini; +42.0106
Da) were searched as variable modications. For the peptides
from the HeLa CMs, two SILAC-based labels (Lys6, + 6.0201
Da) and (Arg10, + 10.0083 Da) were used as variable
modications. For the peptides from the MHCC97H and
MHCC97L cells and CMs, the medium labels were (Lys4, +
4.0251 Da) and (Arg6, + 6.0201 Da), and the heavy labels were
(Lys8, + 8.0142 Da) and (Arg10, + 10.0083 Da). The peptide
and protein identications were ltered by PD to keep the FDR
1%. At least one unique peptide was required for each
protein identication.
Bioinformatic Analysis. The classical secreted proteins
were searched using Signal or Secreted as keywords in
UniProtKB, and the signal peptide was predicted by the SignalP
4.1 server15 (http://www.cbs.dtu.dk/services/SignalP/). The
nonclassical secreted proteins were predicted using a
SecretomeP 2.016 server (http://www.cbs.dtu.dk/services/
SecretomeP/) with an NN score >0.5, but not at a time
predicted to contain a signal peptide. The exosome proteins
were matched by the ExoCarta database17 (http://exocarta.
org/). The biological process annotations and protein
classications were performed using PANTHER18 for GO
analysis (http://pantherdb.org/).
DOI: 10.1021/acs.analchem.6b00910
Anal. Chem. 2016, 88, 49714978
Article
Analytical Chemistry
DOI: 10.1021/acs.analchem.6b00910
Anal. Chem. 2016, 88, 49714978
Article
Analytical Chemistry
(Table S-3), far exceeding the totals from two previous reports
(386 28 or 611 29 proteins), in serum-free CMs from
MHCC97H/MHCC97L cells. Of these 1300 CoPros, 1011
CoPros could be classied as part of the secretome (classical,
nonclassical secreted protein, and extracellular exosome
protein), which includes various growth factors and cytokines
involved in tumor proliferation and metastasis, such as Tgfb1,
Tgfb2, Gpi, Bmp1, Bmp2, Ccl15, Ccl20, Cxcl15, Cxcl16, and
Pf4.
In this data set, 861 CoPros could be reliably quantied in at
least two replicates. A gene ontology (GO) analysis suggests
that several biological processes such as translation, extracellular
matrix disassembly, and signal recognition particle (SRP)dependent cotranslational protein targeting members are
signicantly up-regulated in the CMs from MHCC97H (Figure
4a, Table S-4). Among these samples, several metalloproteases
(Adam9, Adam15, Mmp7) and hydrolases (Ctsb, Ctsd, Ctsl,
DOI: 10.1021/acs.analchem.6b00910
Anal. Chem. 2016, 88, 49714978
Article
Analytical Chemistry
Ctss, Ctsv) involved in extracellular matrix disassembly were
quantied with high expression, and these proteins were
believed to have participated in basement membrane
degradation and been implicated in tumor invasion and
metastasis.30 While other biological processes, such as
regulation of ligase activity, cellular response to stress, and
homeostatic process were signicantly down-regulated in the
CMs from MHCC97H. We detected a series of proteasome
subunits associated with regulation of ligase activity that
exhibited dierent degrees of reduction: Psma1, Psma2,
Psma3, Psma4, Psma5, Psma6, and Psma7 and Psmb1,
Psmb2, Psmb3, Psmb4, Psmb5, Psmb6, Psmb7, and Psmb8.
The informative and quantitative nature of these secretome
data enables an in-depth biological function analysis that may
aid biomarker discovery while helping to explain the
mechanisms of tumor growth, proliferation, and invasion.
Proteins with ratios more than 2 or less than 0.5
(Log2Heavy/Medium >1 or < 1) and t-test P values less
than 0.05 were considered to be signicantly secreted. On this
basis, we found that the levels of 43 and 18 CoPros were
elevated and decreased in the MHCC97H CMs versus those of
MHCC97L, respectively (Figure 4b), including several
cytokines, growth factors, and proteases. Apolipoprotein E
(Apoe), a secreted protein with a key role in lipid binding and
transport, showed the largest increase (14.2-fold) in
MHCC97H CMs; this protein has already demonstrated
overexpression in various cancers, including HCC.31 Granulins
(Grn), a pluripotent growth factor, was up-regulated 2.3-fold in
MHCC97H CMs. The overexpression of granulins implies that
the growth and invasion of HCC are promoted.32 In addition,
some members of the Cathepsin family, such as Cathepsin B
(Ctsb, 2.1-fold), Cathepsin D (Ctsd, 2.3-fold), and Cathepsin S
(Ctss, 3.1-fold), were also up-regulated in the MHCC97H
CMs. These proteins were involved in the disassembly and
organization of the extracellular matrix, which are key steps
during the migration and invasion of tumor cells.33 Protein
NDRG1 (Ndrg1), an important tumor metastasis suppressor in
many cell types,34 showed the largest decrease (0.23-fold) in
the MHCC97H CMs. Although reports indicate that many of
the regulated proteins identied in our data play key roles in
tumor metastasis, most of these studies were performed based
on intracellular proteins. From an extracellular perspective, our
results contain abundant information based on quantitative
proteomics, improving our understanding of tumor invasion
and metastasis.
Quantitative Comparison between Extracellular and
Intracellular Proteomes. Given the signicant dierences
between MHCC97L and MHCC97H regarding cell proliferation and invasion and the shared genetic background of these
two cells, we deduced that both the extracellular and
intracellular proteins played key roles in generating dierentially metastatic potentials. In previous studies of metastatic
tumor cells, most of the attention was focused on either
intracellular or extracellular proteins; few studies35 combined
both. Unfortunately, the above extracellular proteins were
obtained under serum-free conditions, which might perturb the
real status of the cells.
The intracellular proteins extracted from these two SILAClabeled HCCs were also subjected to quantitative proteomic
analyses (Figure 5a, Table S-5). A protein classication analysis
revealed a signicant dierence in the distribution of
functionalities between the extracellular and intracellular
proteins (Figure 5b). The extracellular proteins dominated in
CONCLUSION
In a typical cell culture (1 107 cells, culture for 24 h)
experiment, 2080 g of secreted proteins could be released
into the extracellular medium across dierent cell types.37
Unfortunately, these secreted proteins can be masked by the
4976
DOI: 10.1021/acs.analchem.6b00910
Anal. Chem. 2016, 88, 49714978
Analytical Chemistry
REFERENCES
(1) (a) Lu, P.; Weaver, V. M.; Werb, Z. J. Cell Biol. 2012, 196, 395
406. (b) Meissner, F.; Scheltema, R. A.; Mollenkopf, H.-J.; Mann, M.
Science 2013, 340, 475478.
(2) (a) Brannon-Peppas, L.; Blanchette, J. O. Adv. Drug Delivery Rev.
2012, 64, 206212. (b) Jessani, N.; Liu, Y.; Humphrey, M.; Cravatt, B.
F. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 1033510340.
(3) (a) Stastna, M.; Van Eyk, J. E. Proteomics 2012, 12, 722735.
(b) Makridakis, M.; Vlahou, A. J. Proteomics 2010, 73, 22912305.
(4) (a) Wilhelm, M.; Schlegl, J.; Hahne, H.; Gholami, A. M.;
Lieberenz, M.; Savitski, M. M.; Ziegler, E.; Butzmann, L.; Gessulat, S.;
Marx, H.; et al. Nature 2014, 509, 582587. (b) Uhlen, M.; Fagerberg,
L.; Hallstrom, B. M.; Lindskog, C.; Oksvold, P.; Mardinoglu, A.;
Sivertsson, .; Kampf, C.; Sjostedt, E.; Asplund, A.; et al. Science 2015,
347, 1260419.
(5) (a) Adler, J. J.; Johnson, D. E.; Heller, B. L.; Bringman, L. R.;
Ranahan, W. P.; Conwell, M. D.; Sun, Y.; Hudmon, A.; Wells, C. D.
Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 1736817373. (b) Cooper, S.
FASEB J. 2003, 17, 333340.
(6) Eichelbaum, K.; Winter, M.; Diaz, M. B.; Herzig, S.; Krijgsveld, J.
Nat. Biotechnol. 2012, 30, 984990.
(7) (a) Hinz, F. I.; Dieterich, D. C.; Tirrell, D. A.; Schuman, E. M.
ACS Chem. Neurosci. 2011, 3, 4049. (b) Dieterich, D. C.; Link, A. J.;
Graumann, J.; Tirrell, D. A.; Schuman, E. M. Proc. Natl. Acad. Sci. U. S.
A. 2006, 103, 94829487.
(8) Bagert, J. D.; Xie, Y. J.; Sweredoski, M. J.; Qi, Y.; Hess, S.;
Schuman, E. M.; Tirrell, D. A. Mol. Cell. Proteomics 2014, 13, 1352
1358.
(9) (a) Finoulst, I.; Vink, P.; Rovers, E.; Pieterse, M.; Pinkse, M.; Bos,
E.; Verhaert, P. J. Proteomics 2011, 75, 2333. (b) Colzani, M.;
Waridel, P.; Laurent, J.; Faes, E.; Ruegg, C.; Quadroni, M. J. Proteome
Res. 2009, 8, 47794788.
(10) Anderson, N. L.; Anderson, N. G. Mol. Cell. Proteomics 2002, 1,
845867.
(11) Bandhakavi, S.; Stone, M. D.; Onsongo, G.; Van Riper, S. K.;
Griffin, T. J. J. Proteome Res. 2009, 8, 55905600.
(12) (a) Millioni, R.; Tolin, S.; Puricelli, L.; Sbrignadello, S.; Fadini,
G. P.; Tessari, P.; Arrigoni, G. PLoS One 2011, 6, e19603. (b) Fonslow,
B. R.; Carvalho, P. C.; Academia, K.; Freeby, S.; Xu, T.; Nakorchevsky,
A.; Paulus, A.; Yates, J. R., III J. Proteome Res. 2011, 10, 36903700.
(c) Sabino, F.; Hermes, O.; Egli, F. E.; Kockmann, T.; Schlage, P.;
Croizat, P.; Kizhakkedathu, J. N.; Smola, H.; auf dem Keller, U. Mol.
Cell. Proteomics 2015, 14, 354370.
(13) (a) Di Girolamo, F.; Righetti, P. G.; Soste, M.; Feng, Y.; Picotti,
P. J. Proteomics 2013, 89, 215226. (b) von Toerne, C.; Kahle, M.;
Schafer, A.; Ispiryan, R.; Blindert, M.; Hrabe De Angelis, M.; Neschen,
S.; Ueffing, M.; Hauck, S. M. J. Proteome Res. 2013, 12, 13311343.
(14) (a) Candiano, G.; Dimuccio, V.; Bruschi, M.; Santucci, L.;
Gusmano, R.; Boschetti, E.; Righetti, P. G.; Ghiggeri, G. M.
Electrophoresis 2009, 30, 24052411. (b) Di Girolamo, F.; Boschetti,
E.; Chung, M. C.; Guadagni, F.; Righetti, P. G. J. Proteomics 2011, 74,
589594.
(15) Petersen, T. N.; Brunak, S.; von Heijne, G.; Nielsen, H. Nat.
Methods 2011, 8, 785786.
(16) Bendtsen, J. D.; Jensen, L. J.; Blom, N.; von Heijne, G.; Brunak,
S. Protein Eng., Des. Sel. 2004, 17, 349356.
(17) Mathivanan, S.; Fahner, C. J.; Reid, G. E.; Simpson, R. J. Nucleic
Acids Res. 2012, 40, D1241D1244.
(18) (a) Thomas, P. D.; Campbell, M. J.; Kejariwal, A.; Mi, H.;
Karlak, B.; Daverman, R.; Diemer, K.; Muruganujan, A.; Narechania, A.
Genome Res. 2003, 13, 21292141. (b) Mi, H.; Dong, Q.;
Muruganujan, A.; Gaudet, P.; Lewis, S.; Thomas, P. D. Nucleic Acids
Res. 2010, 38, D204D210.
(19) (a) Rocha, B.; Calamia, V.; Casas, V.; Carrascal, M.; Blanco, F.
J.; Ruiz-Romero, C. J. Proteome Res. 2014, 13, 10451054. (b) Ong,
S.-E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.;
Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376386.
(c) Stiess, M.; Wegehingel, S.; Nguyen, C.; Nickel, W.; Bradke, F.;
Cambridge, S. B. J. Proteome Res. 2015, 14, 32293238.
ASSOCIATED CONTENT
S Supporting Information
*
Article
AUTHOR INFORMATION
Corresponding Author
*E-mail: lihuazhang@dicp.ac.cn.
Notes
ACKNOWLEDGMENTS
This work was supported by National Basic Research Program
of China (2012CB910604), National Natural Science Foundation (21190043), and The Creative Research Group Project by
NSFC (21321064).
4977
DOI: 10.1021/acs.analchem.6b00910
Anal. Chem. 2016, 88, 49714978
Article
Analytical Chemistry
(20) Tran, J. C.; Doucette, A. A. Anal. Chem. 2008, 80, 15681573.
(21) Sharma, R.; Dill, B. D.; Chourey, K.; Shah, M.; VerBerkmoes, N.
C.; Hettich, R. L. J. Proteome Res. 2012, 11, 60086018.
(22) Botelho, D.; Wall, M. J.; Vieira, D. B.; Fitzsimmons, S.; Liu, F.;
Doucette, A. J. Proteome Res. 2010, 9, 28632870.
(23) Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Nat.
Methods 2009, 6, 359362.
(24) (a) Zhang, X.; Wu, H.; Dobson, J. R.; Browne, G.; Hong, D.;
Akech, J.; Languino, L. R.; Stein, J. L.; Stein, G. S.; Lian, J. B. J. Cell.
Biochem. 2015, 116, 20982108. (b) Necula, L. G.; ChivuEconomescu, M.; Stanciulescu, E. L.; Bleotu, C.; Dima, S. O.;
Alexiu, I.; Dumitru, A.; Constantinescu, G.; Popescu, I.; Diaconu, C. C.
J. Gastrointestin. Liver Dis. 2012, 21, 2329.
(25) Wu, C.-C.; Hsu, C.-W.; Chen, C.-D.; Yu, C.-J.; Chang, K.-P.;
Tai, D.-I.; Liu, H.-P.; Su, W.-H.; Chang, Y.-S.; Yu, J.-S. Mol. Cell.
Proteomics 2010, 9, 11001117.
(26) (a) Li, Y.; Tang, Z. Y.; Ye, S. L.; Liu, Y. K.; Chen, J.; Xue, Q.;
Chen, J.; Gao, D. M.; Bao, W. H. World J. Gastroenterol. 2001, 7, 630
636. (b) Cui, J. F.; Liu, Y. K.; Zhang, L. J.; Shen, H. L.; Song, H. Y.;
Dai, Z.; Yu, Y. L.; Zhang, Y.; Sun, R. X.; Chen, J.; Tang, Z. Y.; Yang, P.
Y. Proteomics 2006, 6, 59535961.
(27) Sleeman, J. P. Cancer Metastasis Rev. 2012, 31, 429440.
(28) Yu, Y.; Pan, X.; Ding, Y.; Liu, X.; Tang, H.; Shen, C.; Shen, H.;
Yang, P. Analyst 2013, 138, 45054511.
(29) Li, X.; Jiang, J.; Zhao, X.; Wang, J.; Han, H.; Zhao, Y.; Peng, B.;
Zhong, R.; Ying, W.; Qian, X. PLoS One 2013, 8, e81921e81921.
(30) (a) Gilkes, D. M.; Semenza, G. L.; Wirtz, D. Nat. Rev. Cancer
2014, 14, 430439. (b) Bonnans, C.; Chou, J.; Werb, Z. Nat. Rev. Mol.
Cell Biol. 2014, 15, 786801.
(31) (a) Arzumanyan, A.; Reis, H. M.; Feitelson, M. A. Nat. Rev.
Cancer 2013, 13, 123135. (b) Zhong, D. N.; Ning, Q. Y.; Wu, J. Z.;
Zang, N.; Wu, J. L.; Hu, D. F.; Luo, S. Y.; Huang, A. C.; Li, L. L.; Li, G.
J. Cancer Sci. 2012, 103, 18331838.
(32) (a) Cheung, S. T.; Wong, S. Y.; Leung, K. L.; Chen, X.; So, S.;
Ng, I. O.; Fan, S. T. Clin. Cancer Res. 2004, 10, 76297636. (b) Ho, J.
C.; Ip, Y. C.; Cheung, S. T.; Lee, Y. T.; Chan, K. F.; Wong, S. Y.; Fan,
S. T. Hepatology 2008, 47, 15241532.
(33) (a) Quail, D. F.; Joyce, J. A. Nat. Med. 2013, 19, 14231437.
(b) Gocheva, V.; Wang, H.-W.; Gadea, B. B.; Shree, T.; Hunter, K. E.;
Garfall, A. L.; Berman, T.; Joyce, J. A. Genes Dev. 2010, 24, 241255.
(34) (a) Kovacevic, Z.; Richardson, D. R. Carcinogenesis 2006, 27,
23552366. (b) Sun, J.; Zhang, D.; Bae, D.-H.; Sahni, S.; Jansson, P.;
Zheng, Y.; Zhao, Q.; Yue, F.; Zheng, M.; Kovacevic, Z.; Richardson, D.
R. Carcinogenesis 2013, 34, 19431954.
(35) (a) Calamia, V.; Rocha, B.; Mateos, J.; Fernandez-Puente, P.;
Ruiz-Romero, C.; Blanco, F. J. J. Proteome Res. 2011, 10, 37013711.
(b) Calamia, V.; Fernandez-Puente, P.; Mateos, J.; Lourido, L.; Rocha,
B.; Montell, E.; Verges, J.; Ruiz-Romero, C.; Blanco, F. J. Mol. Cell.
Proteomics 2012, 11, M111.013417.
(36) Zhang, S.; Wu, Q.; Shan, Y.; Sui, Z.; Zhang, L.; Zhang, Y.
Proteomics 2015, 15, 17811788.
(37) (a) Hsu, C.-W.; Yu, J.-S.; Peng, P.-H.; Liu, S.-C.; Chang, Y.-S.;
Chang, K.-P.; Wu, C.-C. J. Proteome Res. 2014, 13, 47964807. (b) Le
Bihan, M.-C.; Bigot, A.; Jensen, S. S.; Dennis, J. L.; RogowskaWrzesinska, A.; Laine, J.; Gache, V.; Furling, D.; Jensen, O. N.; Voit,
T.; Mouly, V.; Coulton, G. R.; Butler-Browne, G. J. Proteomics 2012,
77, 344356.
4978
DOI: 10.1021/acs.analchem.6b00910
Anal. Chem. 2016, 88, 49714978