A Comparison of Methods To Enhance Protein Detection of

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A Comparison of Methods to Enhance Protein Detection of Lipoproteins by Mass Spectrometry Anna Heink, Sean Davidson, Debi K Swertfeger, Long Jason Lu, and Amy Shah J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00270 • Publication Date (Web): 03 Jun 2015 Downloaded from http://pubs.acs.org on June 10, 2015

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Journal of Proteome Research

A Comparison of Methods to Enhance Protein Detection of Lipoproteins by Mass Spectrometry Anna Heink MS 1, W. Sean Davidson PhD2, Debi K Swertfeger 3, L. Jason Lu3, *Amy S Shah MD MS1 1

Department of Pediatrics, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, MLC 7012, Cincinnati, OH 45229-3039, USA 2

Center for Lipid and Arteriosclerosis Science, Department of Pathology and Laboratory Medicine, University of Cincinnati, 2120 East Galbraith Road, Cincinnati, OH 45237-0507, USA 3

Division of Biomedical Informatics, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, MLC 7024, Cincinnati, OH 45229-3039, USA *Corresponding Author/Reprints: Amy S Shah MD MS. Cincinnati Children’s Hospital Medical Center, Division of Endocrinology. 3333 Burnet Ave ML 7012. Cincinnati OH 45229. Email: [email protected]. Phone: 513-636-4744. Fax: 513-696-7486. Anna Heink email: [email protected] W. Sean Davidson e-mail: [email protected] Debi Swertfeger email [email protected] L.J. Lu e-mail: [email protected] Abbreviations: LRA (lipid removal agent), AALS (anionic acid labile surfactant), OSD (organic solvent delipidation), RT (room temperature), AB (ammonium bicarbonate) Running Title: Mass Spectrometer Comparisons Word Count: 4275

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Abstract We sought to develop a new method to more efficiently analyze lipid bound proteins by mass spectrometry using a combination of a lipid removal agent (LRA) that selectively targets lipid bound proteins, and a mass spectrometry compatible detergent, anionic acid labile surfactant (AALS), that is capable of eluting proteins off the LRA. This method was compared to established methods that use the lipid removal agent alone and straight proteomic analysis of human plasma after organic solvent delipidation (OSD). Plasma from healthy individuals was separated by gel filtration chromatography and prepared for mass spectrometry analysis by each of the described methods. The addition of AALS to LRA increased the overall number of proteins detected in both the high and low density lipoprotein (HDL and LDL) size range, the number of peptide counts for each protein, and the overall sequence coverage. Organic solvent delipidation detected the most proteins, though with some decrease in overall protein detection and sequence coverage due to the presence of non-lipid bound proteins. The use of LRA allows for selection and analysis of lipid bound proteins. The addition of a mass spectrometry compatible detergent improved detection of lipid bound proteins from human plasma using LRA. Key Words: lipoproteins, mass spectrometry, detergents, lipids, surfactant


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Introduction Plasma lipoproteins, such as low and high density lipoproteins (LDL and HDL), play important roles in lipid transport and metabolism. The roles of these lipoproteins are largely mediated by the sequence and structure of associated apolipoproteins (apo), apoB for LDL and apoA-I for HDL. Prior to 2000, both LDL and HDL were thought to contain 1400 counts) that monopolize the duty cycle of the mass spectrometer resulting in a lower mass spectrometer signal intensity and loss of protein detection. This idea is further supported by the improved protein detection in the LDL size range, where there were less proteins in general. Third, while not directly a MS data processing or analysis issue, it should be also noted from a work flow standpoint, OSD is a longer and more technically difficult process which requires desalting by dialysis, solubilization of a protein pellet and an organic layer extraction. This was one reason we developed the LRA method initially. However, given the ability to detect more proteins in both the HDL and LDL size range, OSD does offer some advantages and may be ideal for identifying candidate proteins in biological samples. Additionally, OSD may offer improved detection in the LDL size range making it useful to study the LDL proteome, however, spectral counts were too low to definitively conclude this. The advantages and disadvantages of each method are outlined in Table 3. It should be noted that although 70 typical HDL associated proteins were identified by at least one method, some of HDLs well- known proteins including cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) were not detected 13 ACS Paragon Plus Environment


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by any method. Lack of detection is likely because these proteins are lower in abundance and fall below our limit of detection. Similar results have been reported by others (19-23). Thus, the methods presented here are not ideal for quantitation of these low abundance proteins. Conclusions In conclusion, we found that the addition of an MS compatible detergent improves protein detection of lipid bound proteins separated from human plasma by gel filtration when a lipid binding resin was used. While a straight analysis of the fractions without the use of a phospholipid binding agent detected more proteins, the inability to confidently determine the lipidation status of the identified proteins was viewed as a clear disadvantage.

Supplemental Information Files This material is available free of charge via the Internet at http://pubs.acs.org. 1. A Word file that includes Supplementary Figures 1-3 and Supplementary Table 1. 2. An Excel spreadsheet file containing data for all proteins identified and their peptide count distributions across each fraction. Author contribution This manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Acknowledgements: This work was supported by National Institutes of Health Heart Lung and Blood Institute, K23HL118132 to Shah, R01HL67093 and R01HL104136 to Davidson, and R01HL111829 to Lu. Mass Spectrometry data were collected in the UC Proteomics Laboratory on the 5600 + TripleTof system funded in part through an NIH shared instrumentation grant (S10 RR027015-01; KD Greis-PI). Disclosures: None


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References 1. James, R.W., D. Hochstrasser, J.D. Tissot, M. Funk, R. Appel, F. Barja, C. Pellegrini, A.F. Muller, and D. Pometta Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma. J Lipid Res 1988, 29, 1557-1571. 2. Vaisar, T., S. Pennathur, P.S. Green, S.A. Gharib, A.N. Hoofnagle, M.C. Cheung, J. Byun, S. Vuletic, S. Kassim, P. Singh, H. Chea, R.H. Knopp, J. Brunzell, R. Geary, A. Chait, X.Q. Zhao, K. Elkon, S. Marcovina, P. Ridker, J.F. Oram, and J.W. Heinecke Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL. J Clin Invest 2007, 117, 746-756. 3. Heinecke, J.W. The HDL proteome: a marker--and perhaps mediator--of coronary artery disease. J Lipid Res 2009, 50 Suppl, S167-171. 4. Shah, A.S., L. Tan, J.L. Long, and W.S. Davidson Proteomic diversity of high density lipoproteins: our emerging understanding of its importance in lipid transport and beyond. J Lipid Res 2013, 54, 2575-2585. 5. Bancells, C., F. Canals, S. Benitez, N. Colome, J. Julve, J. Ordonez-Llanos, and J.L. Sanchez-Quesada Proteomic analysis of electronegative low-density lipoprotein. J Lipid Res 2010, 51, 3508-3515. 6. Dashty, M., M.M. Motazacker, J. Levels, M. de Vries, M. Mahmoudi, M.P. Peppelenbosch, and F. Rezaee Proteome of human plasma very low-density lipoprotein and low-density lipoprotein exhibits a link with coagulation and lipid metabolism. Thromb Haemost 2014, 111, 518-530. 7. Lindgren, F.T., H.A. Elliott, and J.W. Gofman The ultracentrifugal characterization and isolation of human blood lipids and lipoproteins, with applications to the study of atherosclerosis. J Phys Colloid Chem 1951, 55, 80-93. 8. Gordon, S., A. Durairaj, J.L. Lu, and W.S. Davidson High-Density Lipoprotein Proteomics: Identifying New Drug Targets and Biomarkers by Understanding Functionality. Curr Cardiovasc Risk Rep 4, 1-8. 15 ACS Paragon Plus Environment


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9. Gordon, S.M., J. Deng, A.B. Tomann, A.S. Shah, L.J. Lu, and W.S. Davidson Multi-dimensional coseparation analysis reveals protein-protein interactions defining plasma lipoprotein subspecies. Mol Cell Proteomics 2013, 12, 3123-3134. 10. Gordon, S.M., J. Deng, L.J. Lu, and W.S. Davidson Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography. J Proteome Res 2010, 9, 52395249. 11. Keller, A., A.I. Nesvizhskii, E. Kolker, and R. Aebersold Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 2002, 74, 5383-5392. 12. Nesvizhskii, A.I., A. Keller, E. Kolker, and R. Aebersold A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 2003, 75, 4646-4658. 13. Davidson, W.S., R.A. Silva, S. Chantepie, W.R. Lagor, M.J. Chapman, and A. Kontush Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance to antioxidative function. Arterioscler Thromb Vasc Biol 2009, 29, 870-876. 14. Meng, F., B.J. Cargile, S.M. Patrie, J.R. Johnson, S.M. McLoughlin, and N.L. Kelleher Processing complex mixtures of intact proteins for direct analysis by mass spectrometry. Anal Chem 2002, 74, 2923-2929. 15. Ross, A.R., P.J. Lee, D.L. Smith, J.I. Langridge, A.D. Whetton, and S.J. Gaskell Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant. Proteomics 2002, 2, 928-936. 16. Zeller, M., E.K. Brown, E.S. Bouvier, and S. Konig Use of an acid-labile surfactant as an SDS substitute for gel electrophoresis and proteomic analysis. J Biomol Tech 2002, 13, 1-4. 17. Konig, S., O. Schmidt, K. Rose, S. Thanos, M. Besselmann, and M. Zeller Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: comparative proteomic studies on rat retina and mouse brain. Electrophoresis 2003, 24, 751-756.


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18. Nomura, E., K. Katsuta, T. Ueda, M. Toriyama, T. Mori, and N. Inagaki Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping. J Mass Spectrom 2004, 39, 202-207. 19. Heller, M., D. Stalder, E. Schlappritzi, G. Hayn, U. Matter, and A. Haeberli Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins. Proteomics 2005, 5, 2619-2630. 20. Holzer, M., R. Birner-Gruenberger, T. Stojakovic, D. El-Gamal, V. Binder, C. Wadsack, A. Heinemann, and G. Marsche Uremia alters HDL composition and function. J Am Soc Nephrol 2011, 22, 1631-1641. 21. Holzer, M., P. Wolf, S. Curcic, R. Birner-Gruenberger, W. Weger, M. Inzinger, D. El-Gamal, C. Wadsack, A. Heinemann, and G. Marsche Psoriasis alters HDL composition and cholesterol efflux capacity. J Lipid Res 2012, 53, 1618-1624. 22. Karlsson, H., P. Leanderson, C. Tagesson, and M. Lindahl Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry. Proteomics 2005, 5, 1431-1445. 23. Watanabe, J., C. Charles-Schoeman, Y. Miao, D. Elashoff, Y.Y. Lee, G. Katselis, T.D. Lee, and S.T. Reddy Proteomic profiling following immunoaffinity capture of high-density lipoprotein: association of acute-phase proteins and complement factors with proinflammatory high-density lipoprotein in rheumatoid arthritis. Arthritis Rheum 2012, 64, 1828-1837.



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Figure 1:

Venn diagram of the number of typical HDL associated and non-typical HDL associated proteins detected by mass spectrometry. Number of proteins detected from HDL size range fractions (20-30) among three methods of preparation: lipid removal agent (LRA), lipid removal agent with anionic acid labile surfactant (LRA + AALS), and organic solvent delipidation (OSD).


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Figure 2:

Unadjusted spectral counts for a given HDL associated proteins across gel-filtered plasma fractions in the HDL size range. LRA (black), LRA + AALS (dashed), and OSD (grey) methods.



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Figure 3:

Top ten HDL associated proteins by total spectral counts across the HDL size range detected by MS using LRA, LRA+AALS and OSD.


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Figure 4:

Venn diagram of the number of typical LDL associated and non LDL associated proteins. Proteins detected by mass spectrometry from LDL size range fractions (13-19) by LRA, LRA+AALS, and OSD. .



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Figure 5:

Unadjusted spectral counts for given LDL associated proteins across gel-filtered plasma fractions in the LDL size range. LRA (black), LRA + AALS (dashed), and OSD (grey).


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Figure 6:

Top ten LDL associated proteins by total spectral counts across the LDL size range detected by MS using LRA, LRA+AALS and OSD.



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Table 1: Presence or absence of lowest abundance HDL associated proteins by method. LRA

LRA+AALS

OSD

C4b-binding protein α-chain

-

-

+

Zinc-α-2-glycoprotein

-

-

+

ApoM

-

+

+

ApoF

-

-

+

ApoL1

+

+

+

ApoC-II

+

+

+

Afamin

-

+

+

Glycoprotein phospholipase D

+

+

+

ApoD

-

+

+

Comp. C2

+

+

+

Sum

4

7

10

Symbols indicate the presence (+) or absence (-) of the protein by each method.


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Table 2: Percent (%) sequence coverage of common HDL associated proteins LRA

LRA+AALS OSD

ApoA-I

55.8

62.6

44.9

ApoA-II

69.0

69.0

53.0

ApoA-IV

7.56

20.7

18.2

PON-1

8.5

22.0

3.9

ApoC-II

-

23.8

8.9

ApoC-III

16.2

27.3

27.3

ApoJ

19.6

23.8

19.4

Percent sequence coverage of a given protein in fraction 25. These data were generated using Scaffold (version 4.3.4, Proteome Software Inc., Portland, OR).



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Table 3: Advantages and Limitations of Three Methods to Analyze Lipid Associated Proteins. Method

Advantages

Limitations

LRA

Can be prepared in a 96 well plate

Fewer proteins compared to OSD

No dialysis or desalting needed Selects for lipid-bound proteins LRA+AALS Can be prepared in a 96 well plate

Fewer proteins compared to OSD

No dialysis or desalting needed

Added cost and steps of AALS

Selects for lipid-bound proteins

AALS requires complete lysis prior to MS.

Improved sequence coverage OSD

Greater number of proteins detected

Detects non lipid-bound proteins Dialysis and solubilization required


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LRA 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

Plasma fractions

OSD Method

Lipid binding resin

LRA

LRA + AALS Trypsin & Acid-labile surfactant

Delipidation

Trypsin

Trypsin

Lipid-bound proteins

Delipidated proteins

Elution from LRA

lysis of surfactant at pH 3.0

Trypsin-digested peptides

MS Analysis

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A Comparison of Methods To Enhance Protein Detection of

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