Deep Intact Proteoform Characterization in Human Cell Lysate Using High-pH and Low-pH Reversed-Phase Liquid Chromatography

  • Dahang Yu
  • Zhe Wang
  • Kellye A. Cupp-Sutton
  • Xiaowen Liu
  • Si WuEmail author
Focus: Protein Post-translational Modifications: Research Article


Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low-abundance PTM-modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics.


Top-down proteomics Mass spectrometry Liquid chromatography RPLC Intact proteoforms 



We thank Dr. Anthony Burgett for providing the Hela cells. This work was partly supported by grants from NIH NIAID R01AI141625, NIAID CSGADP Pilot project (NIH 5U01AI101990-04, BRI no. FY15109843), NIH NIGMS R01GM118470, OCAST HR16-125, and OU FIP program.

Supplementary material

13361_2019_2315_MOESM1_ESM.xlsx (344 kb)
ESM 1 (XLSX 344 kb)
13361_2019_2315_MOESM2_ESM.pdf (1.4 mb)
ESM 2 (PDF 1467 kb)


  1. 1.
    Hershko, A., Heller, H., Eytan, E., Kaklij, G., Rose, I.A.: Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown. Proc. Natl. Acad. Sci. 81, 7021–7025 (1984)PubMedCrossRefGoogle Scholar
  2. 2.
    Recht, J., Tsubota, T., Tanny, J., Diaz, R., Berger, J.M., Zhang, X., Garcia, B., Shabanowitz, J., Burlingame, A., Hunt, D.: Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis. Proc. Natl. Acad. Sci. 103, 6988–6993 (2006)PubMedCrossRefGoogle Scholar
  3. 3.
    Grunstein, M.: Histone acetylation in chromatin structure and transcription. Nature. 389, 349 (1997)PubMedCrossRefGoogle Scholar
  4. 4.
    Miao, J., Lawrence, M., Jeffers, V., Zhao, F., Parker, D., Ge, Y., Sullivan Jr., W.J., Cui, L.: Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during P lasmodium falciparum intraerythrocytic development. Mol. Microbiol. 89, 660–675 (2013)PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Phanstiel, D., Brumbaugh, J., Berggren, W.T., Conard, K., Feng, X., Levenstein, M.E., McAlister, G.C., Thomson, J.A., Coon, J.J.: Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells. Proc. Natl. Acad. Sci. 105, 4093–4098 (2008)PubMedCrossRefGoogle Scholar
  6. 6.
    Shen, X., Sun, L.: Systematic evaluation of immobilized trypsin-based fast protein digestion for deep and high-throughput bottom-up proteomics. Proteomics. 18, 1700432 (2018)CrossRefGoogle Scholar
  7. 7.
    Gautier, V., Boumeester, A.J., Lössl, P., Heck, A.J.: Lysine conjugation properties in human IgGs studied by integrating high-resolution native mass spectrometry and bottom-up proteomics. Proteomics. 15, 2756–2765 (2015)PubMedCrossRefGoogle Scholar
  8. 8.
    Pruitt, K.D., Tatusova, T., Maglott, D.R.: NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 35, D61–D65 (2006)PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Hubbard, T.J., Aken, B.L., Beal, K., Ballester, B., Cáccamo, M., Chen, Y., Clarke, L., Coates, G., Cunningham, F., Cutts, T.: Ensembl 2007. Nucleic Acids Res. 35, D610–D617 (2006)PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Melani, R.D., Skinner, O.S., Fornelli, L., Domont, G.B., Compton, P.D., Kelleher, N.L.: Mapping proteoforms and protein complexes from king cobra venom using both denaturing and native top-down proteomics. Mol. Cell. Proteomics. M115, 056523 (2016)Google Scholar
  11. 11.
    Rhoads, T.W., Rose, C.M., Bailey, D.J., Riley, N.M., Molden, R.C., Nestler, A.J., Merrill, A.E., Smith, L.M., Hebert, A.S., Westphall, M.S.: Neutron-encoded mass signatures for quantitative top-down proteomics. Anal. Chem. 86, 2314–2319 (2014)PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Peng, Y., Gregorich, Z.R., Valeja, S.G., Zhang, H., Cai, W., Chen, Y.-C., Guner, H., Chen, A.J., Schwahn, D.J., Hacker, T.A.: Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction. Mol Cell Proteomics. M114, 040675 (2014)Google Scholar
  13. 13.
    Riley, N.M., Sikora, J.W., Seckler, H.S., Greer, J.B., Fellers, R.T., LeDuc, R.D., Westphall, M.S., Thomas, P.M., Kelleher, N.L., Coon, J.J.: The value of activated ion electron transfer dissociation for high-throughput top-down characterization of intact proteins. Analytical chemistry. 90, 8553-8560 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    McCool, E.N., Lubeckyj, R.A., Shen, X., Chen, D., Kou, Q., Liu, X., Sun, L.: Deep top-down proteomics using capillary zone electrophoresis-tandem mass spectrometry: identification of 5700 Proteoforms from the Escherichia coli proteome. Anal. Chem. 90, 5529–5533 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Anderson, L.C., DeHart, C.J., Kaiser, N.K., Fellers, R.T., Smith, D.F., Greer, J.B., LeDuc, R.D., Blakney, G.T., Thomas, P.M., Kelleher, N.L.: Identification and characterization of human proteoforms by top-down LC-21 tesla FT-ICR mass spectrometry. J. Proteome Res. 16, 1087–1096 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Karch, K.R., Coradin, M., Zandarashvili, L., Kan, Z.-Y., Gerace, M., Englander, S.W., Black, B.E., Garcia, B.A.: Hydrogen-deuterium exchange coupled to top-and middle-down mass spectrometry reveals histone tail dynamics before and after nucleosome assembly. Structure. 26, 1651–1663. e1653 (2018)PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Dang, X., Singh, A., Spetman, B.D., Nolan, K.D., Isaacs, J.S., Dennis, J.H., Dalton, S., Marshall, A.G., Young, N.L.: Label-free relative quantitation of isobaric and isomeric human histone H2A and H2B variants by Fourier transform ion cyclotron resonance top-down MS/MS. J. Proteome Res. 15, 3196–3203 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Wu, S., Yang, F., Zhao, R., Tolic, N., Robinson, E.W., Camp, D.G., Smith, R.D., Pasa-Tolic, L.: Integrated workflow for characterizing intact phosphoproteins from complex mixtures. Anal. Chem. 81, 4210–4219 (2009)PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Wang, Z., Liu, X., Muther, J., James, J.A., Smith, K., Wu, S.: Top-down Mass Spectrometry Analysis of Human Serum Autoantibody Antigen-Binding Fragments. Scientific reports. 9, 2345 (2019)Google Scholar
  20. 20.
    Bloh, A.M., Campos, J.M., Alpert, G., Plotkin, S.A.: Determination of N-formimidoylthienamycin concentration in sera from pediatric patients by high-performance liquid chromatography. J. Chromatogr. B Biomed. Sci. Appl. 375, 444–450 (1986)CrossRefGoogle Scholar
  21. 21.
    Yang, Y., Gu, D., Aisa, H.A., Ito, Y.: Studies on the effect of column angle in figure-8 centrifugal counter-current chromatography. J. Chromatogr. A. 1218, 6128–6134 (2011)PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Chen, B., Peng, Y., Valeja, S.G., Xiu, L., Alpert, A.J., Ge, Y.: Online hydrophobic interaction chromatography–mass spectrometry for top-down proteomics. Anal. Chem. 88, 1885–1891 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Wang, Z., Ma, H., Smith, K., Wu, S.: Two-dimensional separation using high-pH and low-pH reversed phase liquid chromatography for top-down proteomics. Int. J. Mass Spectrom. 427, 43–51 (2018)PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Xiu, L., Valeja, S.G., Alpert, A.J., Jin, S., Ge, Y.: Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down proteomics. Anal. Chem. 86, 7899–7906 (2014)PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Zhou, M., Wu, S., Stenoien, D.L., Zhang, Z., Connolly, L., Freitag, M., Paša-Tolić, L.: Profiling Changes in Histone Post-translational Modifications by Top-Down Mass Spectrometry. In: Wajapeyee N, Gupta R (eds.). Springer New York, New York, NY, (2017)Google Scholar
  26. 26.
    Vellaichamy, A., Tran, J.C., Catherman, A.D., Lee, J.E., Kellie, J.F., Sweet, S.M., Zamdborg, L., Thomas, P.M., Ahlf, D.R., Durbin, K.R.: Size-sorting combined with improved nanocapillary liquid chromatography− mass spectrometry for identification of intact proteins up to 80 kDa. Anal. Chem. 82, 1234–1244 (2010)PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Tran, J.C., Zamdborg, L., Ahlf, D.R., Lee, J.E., Catherman, A.D., Durbin, K.R., Tipton, J.D., Vellaichamy, A., Kellie, J.F., Li, M.: Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature. 480, 254–258 (2011)PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Haselberg, R., de Jong, G.J., Somsen, G.W.: Low-flow sheathless capillary electrophoresis–mass spectrometry for sensitive glycoform profiling of intact pharmaceutical proteins. Anal. Chem. 85, 2289–2296 (2013)PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Zhao, Y., Sun, L., Knierman, M.D., Dovichi, N.J.: Fast separation and analysis of reduced monoclonal antibodies with capillary zone electrophoresis coupled to mass spectrometry. Talanta. 148, 529–533 (2016)PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Ansong, C., Wu, S., Meng, D., Liu, X., Brewer, H.M., Kaiser, B.L.D., Nakayasu, E.S., Cort, J.R., Pevzner, P., Smith, R.D.: Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella typhimurium in response to infection-like conditions. Proc. Natl. Acad. Sci. 110, 10153–10158 (2013)PubMedCrossRefGoogle Scholar
  31. 31.
    Wu, S., Brown, R.N., Payne, S.H., Meng, D., Zhao, R., Tolić, N., Cao, L., Shukla, A., Monroe, M.E., Moore, R.J.: Top-down characterization of the post-translationally modified intact periplasmic proteome from the bacterium Novosphingobium aromaticivorans. International journal of proteomics. 2013, 10 (2013)Google Scholar
  32. 32.
    Kelly, R.T., Page, J.S., Tang, K., Smith, R.D.: Array of chemically etched fused-silica emitters for improving the sensitivity and quantitation of electrospray ionization mass spectrometry. Anal. Chem. 79, 4192–4198 (2007)PubMedCrossRefGoogle Scholar
  33. 33.
    Kou, Q., Xun, L., Liu, X.: TopPIC: a software tool for top-down mass spectrometry-based proteoform identification and characterization. Bioinformatics. 32, 3495–3497 (2016)PubMedPubMedCentralGoogle Scholar
  34. 34.
    Guner, H., Close, P.L., Cai, W., Zhang, H., Peng, Y., Gregorich, Z.R., Ge, Y.: MASH suite: a user-friendly and versatile software interface for high-resolution mass spectrometry data interpretation and visualization. J. Am. Soc. Mass Spectrom. 25, 464–470 (2014)PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Fellers, R.T., Greer, J.B., Early, B.P., Yu, X., LeDuc, R.D., Kelleher, N.L., Thomas, P.M.: ProSight Lite: graphical software to analyze top-down mass spectrometry data. Proteomics. 15, 1235–1238 (2015)PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Durbin, K.R., Fornelli, L., Fellers, R.T., Doubleday, P.F., Narita, M., Kelleher, N.L.: Quantitation and identification of thousands of human proteoforms below 30 kDa. J. Proteome Res. 15, 976–982 (2016)PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Cleland, T.P., DeHart, C.J., Fellers, R.T., VanNispen, A.J., Greer, J.B., LeDuc, R.D., Parker, W.R., Thomas, P.M., Kelleher, N.L., Brodbelt, J.S.: High-throughput analysis of intact human proteins using UVPD and HCD on an orbitrap mass spectrometer. J. Proteome Res. 16, 2072–2079 (2017)PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Valeja, S.G., Xiu, L., Gregorich, Z.R., Guner, H., Jin, S., Ge, Y.: Three dimensional liquid chromatography coupling IEC/HIC/RPC for effective protein separation in top-down proteomics. Anal. Chem. 87, 5363 (2015)PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Cai, W., Tucholski, T., Chen, B., Alpert, A.J., McIlwain, S., Kohmoto, T., Jin, S., Ge, Y.: Top-down proteomics of large proteins up to 223 kDa enabled by serial size exclusion chromatography strategy. Anal. Chem. 89, 5467–5475 (2017)PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Gilar, M., Olivova, P., Daly, A.E., Gebler, J.C.: Orthogonality of separation in two-dimensional liquid chromatography. Anal. Chem. 77, 6426–6434 (2005)PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Dwivedi, R.C., Spicer, V., Harder, M., Antonovici, M., Ens, W., Standing, K.G., Wilkins, J.A., Krokhin, O.V.: Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics. Anal. Chem. 80, 7036–7042 (2008)PubMedCrossRefGoogle Scholar
  42. 42.
    Wang, Y., Yang, F., Gritsenko, M.A., Wang, Y., Clauss, T., Liu, T., Shen, Y., Monroe, M.E., Lopez-Ferrer, D., Reno, T.: Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. Proteomics. 11, 2019–2026 (2011)PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Delmotte, N., Lasaosa, M., Tholey, A., Heinzle, E., Huber, C.G.: Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis. J. Proteome Res. 6, 4363–4373 (2007)PubMedCrossRefGoogle Scholar
  44. 44.
    Baghdady, Y.Z., Schug, K.A.: Qualitative evaluation of high pH mass spectrometrycompatible reversed phase liquid chromatography for altered selectivity in separations of intact proteins. Journal of Chromatography A. 1599, 108-114 (2019)PubMedCrossRefGoogle Scholar
  45. 45.
    Schaffer, L.V., Rensvold, J.W., Shortreed, M.R., Cesnik, A.J., Jochem, A., Scalf, M., Frey, B.L., Pagliarini, D.J., Smith, L.M.: Identification and quantification of murine mitochondrial proteoforms using an integrated top-down and intact-mass strategy. J. Proteome Res. 17, 3526–3536 (2018)PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Choi, B., Hercules, D., Gusev, A.: LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices. Fresenius J. Anal. Chem. 369, 370–377 (2001)PubMedCrossRefGoogle Scholar
  47. 47.
    Pagano, A., Rovelli, G., Mosbacher, J., Lohmann, T., Duthey, B., Stauffer, D., Ristig, D., Schuler, V., Meigel, I., Lampert, C.: C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABAB receptors. J. Neurosci. 21, 1189–1202 (2001)PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Kim, J., Petritis, K., Shen, Y., Camp II, D.G., Moore, R.J., Smith, R.D.: Phosphopeptide elution times in reversed-phase liquid chromatography. J. Chromatogr. A. 1172, 9–18 (2007)PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Uechi, T., Tanaka, T., Kenmochi, N.: A complete map of the human ribosomal protein genes: assignment of 80 genes to the cytogenetic map and implications for human disorders. Genomics. 72, 223–230 (2001)PubMedCrossRefGoogle Scholar
  50. 50.
    Rich, B.E., Steitz, J.A.: Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly. Mol. Cell. Biol. 7, 4065–4074 (1987)PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Bargis-Surgey, P., Lavergne, J.P., Gonzalo, P., Vard, C., Filhol-Cochet, O., Reboud, J.P.: Interaction of elongation factor eEF-2 with ribosomal P proteins. Eur. J. Biochem. 262, 606–611 (1999)PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Artero-Castro, A., Perez-Alea, M., Feliciano, A., Leal, J.A., Genestar, M., Castellvi, J., Peg, V., Ramon y Cajal, S., LLeonart, M.E.: Disruption of the ribosomal P complex leads to stress-induced autophagy. Autophagy. 11, 1499–1519 (2015)PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Sidoli, S., Lin, S., Xiong, L., Bhanu, N.V., Karch, K.R., Johansen, E., Hunter, C., Mollah, S., Garcia, B.A.: Sequential window acquisition of all theoretical mass spectra (SWATH) analysis for characterization and quantification of histone post-translational modifications. Mol. Cell. Proteomics. 14, 2420–2428 (2015)PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Kikuchi, G.: The glycine cleavage system: composition, reaction mechanism, and physiological significance. Mol. Cell. Biochem. 1, 169–187 (1973)PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    Koyata, H., Hiraga, K.: The glycine cleavage system: structure of a cDNA encoding human H-protein, and partial characterization of its gene in patients with hyperglycinemias. Am. J. Hum. Genet. 48, 351 (1991)PubMedPubMedCentralGoogle Scholar
  56. 56.
    Fujiwara, K., Okamura-Ikeda, K., Motokawa, Y.: Chicken liver H-protein, a component of the glycine cleavage system. Amino acid sequence and identification of the N epsilon-lipoyllysine residue. J. Biol. Chem. 261, 8836–8841 (1986)PubMedPubMedCentralGoogle Scholar
  57. 57.
    Zhou, Z.H., McCarthy, D.B., O'Connor, C.M., Reed, L.J., Stoops, J.K.: The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes. Proc. Natl. Acad. Sci. 98, 14802–14807 (2001)PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Hermes, F.A., Cronan, J.E.: Scavenging of cytosolic octanoic acid by mutant LplA lipoate ligases allows growth of Escherichia coli strains lacking the LipB octanoyltransferase of lipoic acid synthesis. J. Bacteriol. 191, 6796–6803 (2009)PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Jordan, S.W., Cronan Jr., J.E.: The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein: protein transferase. J. Bacteriol. 185, 1582–1589 (2003)PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Park, M.H., Wolff, E.C.: Hypusine, a polyamine-derived amino acid critical for eukaryotic translation. J. Biol. Chem. 293, 18710–18718 (2018)PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Saini, P., Eyler, D.E., Green, R., Dever, T.E.: Hypusine-containing protein eIF5A promotes translation elongation. Nature. 459, 118 (2009)PubMedPubMedCentralCrossRefGoogle Scholar
  62. 62.
    Cano, V.S., Jeon, G.A., Johansson, H.E., Henderson, C.A., Park, J.H., Valentini, S.R., Hershey, J.W., Park, M.H.: Mutational analyses of human eIF5A-1–identification of amino acid residues critical for eIF5A activity and hypusine modification. FEBS J. 275, 44–58 (2008)PubMedCrossRefPubMedCentralGoogle Scholar
  63. 63.
    Qu, Y., Wu, S., Zhao, R., Zink, E., Orton, D.J., Moore, R.J., Meng, D., Clauss, T.R., Aldrich, J.T., Lipton, M.S.: Automated immobilized metal affinity chromatography system for enrichment of Escherichia coli phosphoproteome. Electrophoresis. 34, 1619–1626 (2013)PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of OklahomaNormanUSA
  2. 2.School of Informatics and ComputingIndiana University-Purdue University IndianapolisIndianapolisUSA

Personalised recommendations