Subscriber access provided by DUESSELDORF LIBRARIES
Article
Sequential Phosphoproteomic Enrichment through Complementary Metal-directed Immobilized Metal Ion Affinity Chromatography Chia-Feng Tsai, Chuan-Chih Hsu, Jo-Nan Hung, Yi-Ting Wang, Wai-Kok Choong, Ming-Yao Zeng, Pei-Yi Lin, Ruo-Wei Hong, Ting-Yi Sung, and Yu-Ju Chen Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Dec 2013 Downloaded from http://pubs.acs.org on December 13, 2013
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Sequential Phosphoproteomic Enrichment through Complementary Metal-directed Immobilized Metal Ion Affinity Chromatography Chia-Feng Tsai1,2,#, Chuan-Chih Hsu2,#, Jo-Nan Hung1, Yi-Ting Wang3,4, Wai-Kok Choong5, Ming-Yao Zeng6, Pei-Yi Lin2,Ruo-Wei Hong6, Ting-Yi Sung5, Yu-Ju Chen1,2,3* 1
Department of Chemistry, National Taiwan University, Taipei, Taiwan
2
Institute of Chemistry, Academia Sinica, Taipei, Taiwan
3
Chemical Biology and Molecular Biophysics Program, Taiwan International
Graduate Program, Institute of Chemistry, Academia Sinica, Taipei, Taiwan 4
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
5
Institute of Information Science, Academia Sinica, Taipei, Taiwan
6
Institute of Chemistry and Biochemistry, National Chung Cheng University, Chia-yi
County, Taiwan #
These two authors contributed equally to this manuscript
*Corresponding Author Email Address:
[email protected] Address: 128 Academia Road, Sec. 2, Nankang Taipei, Taiwan 115 Phone Number: +886-2-2789-8660 Fax Number: +886-2-2789-8534
Keywords: Metal-directed immobilized metal ion affinity chromatography; Mass Spectrometry; Phosphoproteomics; Phosphorylation; cancer tissue;
1
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Abbreviations: AA: Acetic acid DHB: 2,5-dihydroxybenzoic acid FDR: False discovery rate; MD-score: Mascot delta score MOC: Metal oxide chromatography UPLC: Ultra performance liquid chromatography FLR: False localization rate PSM: A peptide-spectrum match, with an associated score
2
ACS Paragon Plus Environment
Page 2 of 31
Page 3 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Abstract Methodologies to enrich heterogeneous types of phosphopeptides are critical for comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinct binding affinities of Ga3+ and Fe3+ for phosphopeptides, we designed a metal-directed immobilized metal ion affinity chromatography for the sequential enrichment of phosphopeptides. In Raji B cells, the sequential Ga3+-Fe3+-IMAC strategy displayed a 1.5-3.5-fold superior phosphoproteomic coverage compared to single IMAC (Fe3+, Ti4+, Ga3+ and Al3+). In addition, up to 92% of the 6,283 phosphopeptides were uniquely enriched in either the 1st Ga3+-IMAC (41%) or 2nd Fe3+-IMAC (51%). The complementary properties of Ga3+ and Fe3+ were further demonstrated through the exclusive enrichment of almost all of 1,214 multiply phosphorylated peptides (99.4%) in the Ga3+-IMAC, whereas only 10% of 5,069 monophosphorylated phosphopeptides were commonly enriched in both fractions. The application of sequential Ga3+-Fe3+-IMAC to human lung cancer tissue allowed the identification of 2,560 unique phosphopeptides with only 8% overlap. In addition to the abovementioned mono and multiply phosphorylated peptides, this fractionation ability was also demonstrated on the basic and acidic phosphopeptides: acidophilic phosphorylation sites were predominately enriched in the 1st Ga3+-IMAC (72%), while Pro-directed (85%) and basophilic (79%) phosphorylation sites were enriched in the 2nd Fe3+-IMAC. This strategy provided complementary mapping of different kinase substrates in multiple cellular pathways related to cancer invasion and metastasis of lung cancer. Given the fractionation ability and ease of tip preparation of this Ga3+-Fe3+-IMAC, we propose that this strategy allows more comprehensive characterization of the phosphoproteome both in vitro and in vivo.
3
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 31
Introduction Dynamic phosphorylation induces alterations of protein conformations and modulates a variety of molecular and cellular functions
1,2
. Mass spectrometry
(MS)-based strategies3 are utilized for qualitative and quantitative characterization of spatial and temporal changes in phosphorylation events
4,5
. Although MS technology
has been commonly used for phosphorylation sites identification in complex phosphoproteome, achieving comprehensive coverage of the human phosphorylation network, such as in human surgical tissue specimens, remains a challenge. The complexity of the dynamic kinase-substrate network clearly necessitates the development
of
systems
biology
approaches
to
delineate
the
phosphorylation-mediated signal propagation and the defects associated with disease3. For example, protocols for the large-scale in vivo investigation of the phosphoproteomes of mammalian tissues are constantly being developed and applied6,7. Though multiple public resource databases document these data, such as PhosphoELM, HPRD 9.0, phosphositeplus (version 20121203), sysPTM 1.1 and UniProt8-12, however, only approximately 1,200 phosphoproteins have been reported as kinase substrates (Supplementary Table 1). Immobilized metal affinity chromatography (IMAC)13,14 and metal oxide chromatography (MOC)15,16 are widely used for enriching phosphopeptides. Their enrichment specificity is greatly improved17-20 and various enrichment strategies have been reported to allow the identification of thousands of phosphorylation sites21,22. However, the physicochemical properties of phosphopeptides critically determine their sequence-dependent ionization efficiency. For example, the detection of multiply phosphorylated peptides may be suppressed by the presence of monophosphorylated peptides with a relatively more positive charge21,23. Besides, the substrates of Ser/Thr protein kinases, which can be categorized into Pro-directed, basophilic and 4
ACS Paragon Plus Environment
Page 5 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
acidophilic groups24, display different ionization efficiencies. Therefore, it is necessary to develop approaches for fractionation of different types of phosphopeptides. To address the above issues, many groups used a tandem purification strategy to improve the identification coverage of the phosphoproteome. Thingholm et al. reported the SIMAC (sequential elution from IMAC) strategy for separating multiply phosphorylated peptides from monophosphorylated peptides based on acid and base elution through Fe3+-IMAC, followed by TiO2 enrichment23. 16% overlapping phosphopeptides (n=68) have been reported between the acid and base fractions. Most multiply phosphorylated peptides (96%) have been found in the base elution fraction. Choi et al. used sequential Fe3O4/TiO2 for phosphopeptides purification from L6 muscle cell lysates. Multiply phosphorylated peptides initially purified by magnetic Fe3O4. In the following, remaining phosphorylated peptides were captured on TiO2 25. Besides, Ye and colleagues reported the IMAC-IMAC strategy, which controls the bed volume of Fe3+-IMAC resin26. The overlap of phosphopeptides between the first (n=885) and second IMAC (n=793) was found to be 12% (n=183). Extensive fractionation through ion-exchange chromatography is another popular approach. Because phosphopeptides containing multiple basic residues will carry higher charge than common tryptic peptides, they are more difficult to be enriched via IMAC21. To address this issue, Hennrich et al. employed a tandem strong cation exchange (SCX) 5
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
chromatography for the enrichment of basic phosphopeptides. This approach resulted in a nearly 20-fold increase in the number of identified basic phosphopeptides compared to single SCX analysis27. Following phosphopeptide purification via Ti-IMAC, Dong et al. performed strong anion exchange (SAX) chromatography to remove acidophilic substrates; thus, the detection ability of basophilic substrates was increased28. The success of this strategy was demonstrated by the 16.8% overlapping phosphopeptides detected between the depleted fraction and the SAX-bound fraction. These findings indicate that each strategy has intrinsic biases in terms of capturing phosphopeptides of a specific nature and suggest that integrating different methods can separate diverse phosphopeptides. Transition metal ions with multiple coordination orbitals are the best choice for use in the IMAC. Trivalent or tetravalent transition metal ions, such as Ti4+, Fe3+ and Zr4+ were reported to enrich complementary phosphopeptides29-31. These studies adopted a parallel enrichment procedure, rather than conducting sequential enrichment, to increase identification coverage, which was most likely due to the incompatibility of buffer conditions between the methods. For example, Ti4+ provide more electrophilic orbitals, leading to a stronger coordination affinity for phosphopeptides compared to Fe3+ 30, which complements the Fe3+-IMAC method for enriching acidic and basic phosphopeptides31. However, compared to other metal ions, Ti4+ ions display more unoccupied valence orbitals, leading to stronger coordination with phosphate groups as well as carboxylic acid groups. Thus, higher organic contents and more acidic conditions must be applied to provide better purification 6
ACS Paragon Plus Environment
Page 6 of 31
Page 7 of 31
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
specificity31. However, such conditions will reduce the binding of phosphates to other metal ions. Thus, it is difficult to couple Ti4+-IMAC with the use of other metal ions for sequential purification. In addition to these commonly used transition metal ions, Ga3+ ions have been employed for phosphopeptide purification32,33 due to the similarity of their ionic radii, charge and coordination preferences to Fe3+ ions. Ga3+-IMAC has been shown to purify less mono- but more multiply phosphorylated peptides than Fe3+-IMAC33,34. Interestingly, although both Fe3+ and Ga3+ show high specificity for phosphopeptides, it has been observed that the Ga3+-loaded agarose binds more weakly to multiphosphorylated peptides compared to Fe3+-loaded agarose34. These findings imply that Ga3+ exhibits a weaker affinity for phosphate groups. We therefore hypothesized that a sequential phosphopeptide enrichment approach could be designed involving two-metal ion-directed IMAC performed under comparable buffer conditions to enrich phosphopeptides with different sequences or natures. In this work, we present a Ga3+-Fe3+-IMAC method for the sequential purification of phosphopeptides with different properties. We hypothesized that in Ga3+-IMAC, weaker metal ions are only able to retain phosphopeptides with more negative charges, i.e., multiply phosphorylated and acidophilic groups. Fe3+-IMAC exhibits stronger metal-phosphate coordination, which may provide a sufficient affinity to enrich the phosphopeptides with monophosphorylated sites, basophilic and Pro-directed residues. Using Raji B cells, the sequential Ga3+-Fe3+-IMAC method displayed a much better detection sensitivity compared to the use of IMAC alone; a low percentage of overlapping identification results (
