Subscriber access provided by United Arab Emirates University | Libraries Deanship
Letter
Hydrophobic tagging-assisted N-termini enrichment (HYTANE) for in-depth N-terminome analysis Lingfan Chen, Yichu Shan, Yejing Weng, Zhigang Sui, Xiaodan Zhang, Zhen Liang, Lihua Zhang, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02453 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 22, 2016
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 6
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
Hydrophobic tagging-assisted N-termini enrichment (HYTANE) for in-depth N-terminome analysis Lingfan Chena,b,c, Yichu Shana,c, Yejing Wenga,b, Zhigang Suia, Xiaodan Zhanga, Zhen Lianga, Lihua Zhanga,*, Yukui Zhanga a Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China b University of the Chinese Academy of Sciences, Beijing 100049, China c These authors contributed equally ABSTRACT: The analysis of protein N-termini is of great importance for understanding the protein function and elucidating the proteolytic processing. Herein, we develop a negative enrichment strategy, termed as hydrophobic tagging-assisted N-termini enrichment (HYTANE) to achieve a global N-terminome analysis. The HYTANE strategy showed a high efficiency in hydrophobic tagging and C18 material-assisted depletion using bovine serum albumin (BSA) as the sample. Furthermore, this strategy was also applied to N-termini profiling from S. cerevisiae cell lysates and enabled the identification of 1,096 protein N-termini, representing the largest N-terminome dataset of S. cerevisiae. The identified N-terminal peptides accounted for 99% of all identified peptides and no deficiency in acidic, histidine (His)-containing and His-free N-terminal peptides was observed. The presented HYTANE strategy is therefore a highly selective, efficient and unbiased strategy for the large scale N-terminome analysis. Furthermore, using the HYTANE strategy we identified 329 cleavage sites and 291 substrates of caspases in Jurkat cells, demonstrating the great promise of HYTANE strategy for protease research. Data are available via ProteomeXchange with identifier PXD004690.
Protein N-terminal sequences and modifications play significant roles in protein targeting, protein stability, complex formation, and so forth1-5. In addition, the determination of protein N-termini not only allows evidence-based gene annotation6-9, but also helps to elucidate proteolytic processing events10-16. Therefore, the characterization of N-termini has drawn increasing attention in recent years. However, the highly dynamic nature of proteomes as well as abundant non-Nterminal peptides hinders the deep analysis of N-termini. Therefore, the enrichment of N-terminal peptides prior to MS analysis is indispensable. Due to the inherent nature of positive selection, naturally modified N-termini, for example, protein N-terminal acetylation, which is widespread (85%) in many eukaryotic cells10, cannot be enriched by positive selection6,12,17-19. In negative selection approaches, amines are blocked at the protein level, followed by digestion to expose newly free amines for internal peptides. Through the capture of free amines, internal peptides can be depleted, thereby enabling enrichment of both free and naturally modified N-termini. Amine reactive scavenger materials, including Nhydroxysuccinimide (NHS)-activated Sepharose20, cyanogen bromide (CNBr)-activated Sepharose21, isocyanate resin22, aldehyde functionalized beads (POROS-AL)23, and so on24,25, have been employed for the depletion of internal peptides. However, due to the limited efficiency of solid-liquid reaction, the removal of internal peptides is generally incomplete. To circumvent the above-mentioned problem, a series of methods based on liquid-liquid reaction have been proposed. In these methods, amine reactive reagents were adopted to incorporate affinity tags, such as biotin, phosphate group and
sulfhydryl group, to internal peptides. The tagged peptides can be depleted by the corresponding affinity materials, including immobilized avidin beads26, TiO227 and gold nanoparticles28. These methods are capable of depleting internal peptides with high efficiency. However, a large amount of scavenger materials are required for removal of internal peptides, which might decrease the recovery of N-terminal peptides. Terminal amine isotopic labeling of substrates (TAILS)29, however, used a home-synthesized aldehyde polymer, enabling the efficient depletion of internal peptides with low nonspecific adsorption due to the high content of aldehyde groups and watersolubility of aldehyde polymer. Alternatively, combined fractional diagonal chromatography (COFRADIC) was developed to isolate N-terminal peptides without introducing scavenger material30. In COFRADIC, peptides are fractionated using reversed-phase (RP) separation, following which hydrophobic trinitrophenyl groups are introduced to internal peptides. After each fraction is subjected to an additional round of RP separation, the trinitrophenylated internal peptides are expected to elute late, and would therefore be segregated from N-terminal peptides. A variation of COFRADIC, termed charge-based fractional diagonal chromatography(ChaFRADIC)31, using strong cation exchange (SCX) separation and charge-reduced labeling to isolate the N-terminal peptides from internal peptides. However, in cases of fractional diagonal chromatography-based methods, the extensive fractionation and desalting steps make it time- and labor-consuming, and may induce significant sample loss. Instead, Lai et al.32 proposed a charge-reversal approach, in which internal peptides derivatized with negatively charged sulfonate groups lack the ability to bind to
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 2 of 6
Scheme 1. Flow chart for negative enrichment of N-terminal peptides by HYTANE strategy.
SCX and thus could be effectively removed by a single SCX separation. The charge- reversal approach dramatically simplified the separation protocol for N-terminal peptides enrichment. However, owing to the lack of positive charge at Nterminal α-amino groups, histidine (His)-free acetylated Ntermini tend to be eluted in the flowthrough fraction of SCX30,33,34. Therefore, the loss of a portion of acetylated Ntermini by charge-reversal approach might be unavoidable. Herein, we propose a facile negative enrichment method, termed as hydrophobic tagging-assisted N-termini enrichment (HYTANE) for N-termini analysis. As shown in Scheme 1, firstly, formaldehyde-based dimethylation is employed to block free amines on both protein N-termini and side chains of lysine. Secondly, proteins are digested with trypsin, generating N-terminal peptides with blocked amines, and internal peptides containing free amines at N-termini. Subsequently, two long alkyl chains are conjugated to free amines using Schiff base reduction reaction. Benefiting from the alkyl chains, internal peptides exhibit significantly enhanced hydrophobicity and thus can be depleted through strong binding onto C18 materials, leaving exclusively N-terminal peptides to be facilely eluted for LC-MS/MS analysis. The efficiency of reductive dimethylation is critical to recover N-terminal peptides because the non-dimethylated Nterminal peptides would be codepleted in the following steps. Besides, the dimethylation also introduces the tag to validate the genuine neo-N-termini. The incorporated tag on N-termini indicates that the neo-N-termini are derived from endogenous proteolysis rather than cleavage during the sample pretreatment35. To assess the dimethylation efficiency, BSA was dimethylated at protein level. After employing a bottom-up analysis of dimethylated BSA, we observed that 93% of the amine-containing sites on proteins were blocked by dimethylation based on peptide-spectrum matches (PSMs)17,31, indicating that the dimethylation at protein level was almost complete.
Then, we evaluated the influence of alkyl chain length on peptide retention on C18 materials. Three types of aminereactive reagents, including octanal, undecanal and hexadecanal were chosen to introduce alkyl chains to a synthetic peptide (RVYVHPI). As shown in Figure S1, 20% ACN was able to completely elute native peptide from C18 trap column, whereas, bis-octylated and bis-undecylated peptides could not elute from the column until the concentration of ACN reached 60% and 80%, respectively. Furthermore, the hydrophobic interaction between C18 group and bis-hexadecyl chains was strong enough to keep the bis-hexadecylated peptides uneluted during the stringent washing step. To maximize the difference in retention between native and tagged peptides, hexadecanal, which exhibited the strongest hydrophobicity, was used to label internal peptides in our following experiment. High efficiency in bis-hexadecyl labeling and C18 material assisted depletion of internal peptides is critical to minimize copurification of internal peptides in the final sample mixture. To examine the efficiency of bis-hexadecyl tagging and C18 material assisted depletion, bovine serum albumin (BSA) tryptic digests, acted as mixed peptides with different physicochemical property, were employed for evaluation. Firstly, MALDI-TOF MS was used for the analysis of bis- hexadecylated BSA digests. As shown in Figure S2, the bis-hexadecyl tagging was nearly complete for different peptides, as indicated by the disappearance of native peptides and monohexadecylated counterparts, and the appearance of signals corresponding to bis-hexadecylated counterparts. In addition, the chromatographic approach was also employed to evaluate the efficiency of bis-hexadecyl labeling. As shown in Figure S3, after bis-hexadecyl labeling and RP C18 separation, the chromatographic peaks of original BSA digests were almost disappeared, revealing most of BSA digests were bishexadecyl labeled (97%). The bis-hexadecylated BSA digests were loaded onto a C18 trap column, followed by washing with 80% ACN. MALDI-TOF MS analysis showed that no
ACS Paragon Plus Environment
Page 3 of 6
Analytical Chemistry Table 1. The number of identified protein N-termini of S. cerevisiae with detailed analysis conditions in different methods
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
Starting material
MS analysis time
(µg)
(min ×fraction)
PTAG27
100
160×6
LTQ-Orbitrap XL
379
ChaFRADIC31
100
127×5
Q-Exactive
496
COFRADIC8
1000
35×120
LTQ-Orbitrap XL
733
HYTANE
100
85×7
Q-Exactive
879
Method
Instrument
Number of protein N-terminia
a
The N-terminal peptides not only fulfil the Arg-C cleavage at their C-termini, but also contain dimethyl or acetyl group on their N-termini, which ensure the high confidence of the results.
Figure 1. MALDI-TOF MS spectra of the tryptic digests of dimethylated bovine serum albumin (BSA). (a) Direct analysis. (b) After treatment by HYTANE strategy. The N-terminal peptide is marked with asterisk.
obvious signals were detected in the eluate (Figure S2). It is worth noting that after the treatment by bis-hexadecyl labeling, peptides with high hydrophilicity (GRAVY < -2) also bound strongly on the C18 trap column during washing with 80% ACN. The results confirmed the high depletion efficiency of C18 material for bis-hexadecylated peptides. In addition, the recovery of N-terminal peptides in HYTANE strategy was evaluated according to previously reports22,36. Light-dimethylated BSA tryptic digests were used as blocked N-termini for evaluation (see experimental section). As shown in Figure S4, by using 23 different peptides, the average recovery was calculated to be 88 ± 1.7% (n=3), based on the peak intensity ratios of the light labeled to the heavy labeled peptides. The high recovery should be contributed to the low sample loss during the sequential bis-hexadecyl tagging and C18 material assisted depletion without extra cleanup, as well as the low non-specific interaction of peptides with C18 material under stringent washing condition. With the high blocking efficiency, high depletion efficiency and high recovery, HYTANE strategy was used to enrich protein N-termini employing BSA as a model system. As shown in Figure 1, before enrichment, many internal peptide peaks with high intensity were observed, largely masking the detection of N-terminal peptides. After the treatment by HYTANE strategy, the peak corresponding to N-terminal peptides was exclusively detected, demonstrating the high selectivity of HYTANE strategy for the enrichment of protein N-termini.
HYTANE strategy was further applied to analyse the Nterminome from S. cerevisiae cell lysates. An enriched fraction corresponding to 10 µg of the original sample was submitted to nanoRPLC-ESI-MS/MS analysis. As a result, 515 Nterminal peptides, corresponding to 494 protein N-termini were identified in three technical replicates, which accounted for 99% of all identified peptides. Only 5 and none of the 520 peptides were identified as unlabeled and hexadecylated peptides, respectively (Table S1), indicating the nearly complete hydrophobic tagging and C18 material assisted depletion in complex samples. To further achieve an in-depth N-terminome profiling of S. cerevisiae cell lysates, three parallel enrichments using 100 µg of S. cerevisiae cell lysates each were performed, followed by nanoSCX-RPLC-ESI-MS/MS analysis. After the strict dataset searching, 920 ± 51 N-terminal peptides were identified with FDR less than 0.5%, corresponding to 879 ± 49 protein Ntermini. Over 63% of the N-termini could be repeatedly identified in three independent experiments, revealing the high reproducibility of the HYTANE strategy. Combining the results from triplicate analysis, a total of 1,096 protein N-termini were identified with 1,156 N-terminal peptides matching (Table S2). The above results were further compared with those previously reported for S. cerevisiae N-terminome analysis. As seen in Table 1, we obtained the largest N-terminome dataset of S. cerevisiae to date. In addition, as shown in Figure 2a, 63%, 74% and 54% of the protein N-termini identified by PTAG, ChaFRADIC, and COFRADIC could be included in the dataset of HYTANE (Figure 2a). Besides, a notable improvement in identification of low-abundance protein Ntermini (3)29,38. 329 (70%) followed Asp (D) in corresponding protein sequences (Figure 3a), as would be expected for caspase-like cleavage products. A sequence logo analysis of the 329 neo-N-termini revealed that the most frequent residues at the P4, P3, P2, and P1’ positions of the cleavage site were Asp (D), Glu (E), Val (V), and Gly (G), respectively (Figure 3b), which was nearly identical to the caspases recognition motifs reported previously12,39. The 329 cleavage sites could be mapped to 291 substrates. A comparison of these substrates with the CASBAH database, which contained a comprehensive list of the caspases substrates (725, Table S4) identified by numerous studies, revealed that our dataset covered a substantial part of the known substrates (145) while discovering a large set of novel substrates (146). Together, these results demonstrated the great potential of our strategy in mapping protease substrates and cleavage sites. The detailed information could be found in Tables S5.
Figure 3. Discovery of caspase cleavage sites and substrates in Jurkat cells. (a) Frequencies of amino acid at P1 position corresponding to the up-regulated neo-N-termini in apoptosis cells. Data are represented as mean ± SD. (b) Sequence logo representation of the frequency of amino acid residues in the caspase-like cleavage sites. Consensus sequences were generated using iceLogo40.
In conclusion, the HYTANE strategy was developed to provide a facile and effective N-terminal enrichment technique. The use of highly reactive hydrophobic tagging reagent, in the
ACS Paragon Plus Environment
Page 5 of 6
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
combination with C18 material-assisted depletion, provided a robust approach for highly selective enrichment of protein Ntermini. Besides, due to the straightforward procedure without extensive fractionation and clean-up steps, and the direct depletion during sample desalting without extra scavenger material, such a strategy showed the merits of high efficiency and no bias in N-terminome analysis. Combined with quantification method (SILAC), such a strategy was successfully applied to identify substrate and cleavage site of caspases. It is anticipated that with the combination of gel-based separation32,38,41, a more in-depth profiling and orthogonal verification of protease cleavage products would be obtained. We expect that this strategy will facilitate the understanding of proteolysis processes, N-terminal modifications and gene annotation.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Author *Phone: +86-411-84379720. Fax: +86-411-84379720. E-mail:
[email protected].
ACKNOWLEDGEMENT The authors are grateful for the financial support from National Basic Research Program of China (2012CB910601) and National Natural Science Foundation (21475127, 21235005).
REFERENCES (1) Scott, D. C.; Monda, J. K.; Bennett, E. J.; Harper, J. W.; Schulman, B. A. Science 2011, 334, 674-678. (2) Shemorry, A.; Hwang, C.-S.; Varshavsky, A. Mol. Cell 2013, 50, 540-551. (3) Shih, Y.-P.; Chou, C.-C.; Chen, Y.-L.; Huang, K.-F.; Wang, A. H.-J. PLoS One 2014, 9, e94812. (4) Lai, Z. W.; Petrera, A.; Schilling, O. Curr. Opin. Chem. Biol. 2015, 24, 71-79. (5) Gawron, D.; Ndah, E.; Gevaert, K.; Van Damme, P. Mol. Syst. Biol. 2016, 12, 858. (6) Bland, C.; Bellanger, L.; Armengaud, J. J. Proteome Res. 2013, 13, 668-680. (7) Lange, P. F.; Huesgen, P. F.; Nguyen, K.; Overall, C. M. J. Proteome Res. 2014, 13, 2028-2044. (8) Helsens, K.; Van Damme, P.; Degroeve, S.; Martens, L.; Arnesen, T.; Vandekerckhove, J.; Gevaert, K. J. Proteome Res. 2011, 10, 3578-3589. (9) Fortelny, N.; Pavlidis, P.; Overall, C. M. Proteomics 2015, 15, 2547-2552. (10) Rogers, L. D.; Overall, C. M. Mol. Cell. Proteomics 2013, 12, 3532-3542. (11) Prudova, A.; Serrano, K.; Eckhard, U.; Fortelny, N.; Devine, D. V.; Overall, C. M. Blood 2014, e49-60. (12) Mahrus, S.; Trinidad, J. C.; Barkan, D. T.; Sali, A.; Burlingame, A. L.; Wells, J. A. Cell 2008, 134, 866-876. (13) Vögtle, F.-N.; Wortelkamp, S.; Zahedi, R. P.; Becker, D.; Leidhold, C.; Gevaert, K.; Kellermann, J.; Voos, W.; Sickmann, A.; Pfanner, N. Cell 2009, 139, 428-439. (14) Schlage, P.; Egli, F. E.; Nanni, P.; Wang, L. W.; Kizhakkedathu, J. N.; Apte, S. S.; auf dem Keller, U. Mol. Cell. Proteomics 2014, 13, 580-593.
(15) Wildes, D.; Wells, J. A. Proc. Natl. Acad. Sci. USA 2010, 107, 4561-4566. (16) Marino, G.; Eckhard, U.; Overall, C. M. ACS Chem. Biol. 2015, 10, 1754-1764. (17) Kim, J.-S.; Dai, Z.; Aryal, U. K.; Moore, R. J.; Camp, D. G.; Baker, S. E.; Smith, R. D.; Qian, W.-J. Anal. Chem. 2013, 85, 6826-6832. (18) Yamaguchi, M.; Nakazawa, T.; Kuyama, H.; Obama, T.; Ando, E.; Okamura, T.-a.; Ueyama, N.; Norioka, S. Anal. Chem. 2005, 77, 645-651. (19) Xu, G.; Shin, S. B. Y.; Jaffrey, S. R. Proc. Natl. Acad. Sci. USA 2009, 106, 19310-19315. (20) McDonald, L.; Beynon, R. J. Nat. Protoc. 2006, 1, 17901798. (21) Zhang, X.; Ye, J.; Højrup, P. J. Proteomics 2009, 73, 240251. (22) Mikami, T.; Takao, T. Anal. Chem. 2007, 79, 7910-7915. (23) Shen, P.-T.; Hsu, J.-L.; Chen, S.-H. Anal. Chem. 2007, 79, 9520-9530. (24) Kuyama, H.; Sonomura, K.; Nishimura, O.; Tsunasawa, S. Anal. Biochem. 2008, 380, 291-296. (25) Zhao, L.; Zhang, Y.; Wei, J.; Cao, D.; Liu, K.; Qian, X. Proteomics 2009, 9, 4416-4420. (26) McDonald, L.; Robertson, D. H.; Hurst, J. L.; Beynon, R. J. Nat. Methods 2005, 2, 955-957. (27) Mommen, G. P.; van de Waterbeemd, B.; Meiring, H. D.; Kersten, G.; Heck, A. J.; de Jong, A. P. Mol. Cell. Proteomics 2012, 11, 832-842. (28) Li, L.; Wu, R.; Yan, G.; Gao, M.; Deng, C.; Zhang, X. Anal. Bioanal. Chem. 2016, 408, 441-448. (29) Kleifeld, O.; Doucet, A.; auf dem Keller, U.; Prudova, A.; Schilling, O.; Kainthan, R. K.; Starr, A. E.; Foster, L. J.; Kizhakkedathu, J. N.; Overall, C. M. Nat. Biotechnol. 2010, 28, 281-288. (30) Staes, A.; Van Damme, P.; Helsens, K.; Demol, H.; Vandekerckhove, J.; Gevaert, K. Proteomics 2008, 8, 1362-1370. (31) Venne, A. S.; Vögtle, F.-N.; Meisinger, C.; Sickmann, A.; Zahedi, R. P. J. Proteome Res. 2013, 12, 3823-3830. (32) Lai, Z. W.; Gomez‐Auli, A.; Keller, E. J.; Mayer, B.; Biniossek, M. L.; Schilling, O. Proteomics 2015, 15, 2470-2478. (33) Van Damme, P.; Staes, A.; Bronsoms, S.; Helsens, K.; Colaert, N.; Timmerman, E.; Aviles, F. X.; Vandekerckhove, J.; Gevaert, K. Nat. Methods 2010, 7, 512-515. (34) Chen, S.-H.; Chen, C.-R.; Chen, S.-H.; Li, D.-T.; Hsu, J.-L. J. Proteome Res. 2013, 12, 3277-3287. (35) Wang, H.; Tang, H.-Y.; Tan, G. C.; Speicher, D. W. J. Proteome Res. 2011, 10, 4993-5005. (36) Xu, P.; Duong, D. M.; Peng, J. J. Proteome Res. 2009, 8, 3944-3950. (37) Emanuelsson, O.; Brunak, S.; von Heijne, G.; Nielsen, H. Nat. Protoc. 2007, 2, 953-971. (38) Dix, M. M.; Simon, G. M.; Wang, C.; Okerberg, E.; Patricelli, M. P.; Cravatt, B. F. Cell 2012, 150, 426-440. (39) Stoehr, G.; Schaab, C.; Graumann, J.; Mann, M. Mol. Cell. Proteomics 2013, 12, 1436-1450. (40) Colaert, N.; Helsens, K.; Martens, L.; Vandekerckhove, J.; Gevaert, K. Nat. Methods 2009, 6, 786-787. (41) Dix, M. M.; Simon, G. M.; Cravatt, B. F. Cell 2008, 134, 679-691.
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
For Table of Contents Only
ACS Paragon Plus Environment
Page 6 of 6
