, Volume 82, Issue 1, pp 101–110 | Cite as

High-Resolution Nano-Liquid Chromatography with Tandem Mass Spectrometric Detection for the Bottom-Up Analysis of Complex Proteomic Samples

  • Magali Dams
  • José Luís Dores-Sousa
  • Robert-Jan Lamers
  • Achim Treumann
  • Sebastiaan EeltinkEmail author
Part of the following topical collections:
  1. 50th Anniversary Commemorative Issue


Liquid chromatography coupled with mass spectrometric detection is one of the major technologies used for protein sequencing, identification, and quantification. This review provides an introduction of the current state-of-the-art technology in peptide profiling using nano-liquid chromatography–mass spectrometry applied to large-scale protein or proteome analysis. In particular, different aspects of the bottom-up proteomics workflow are covered, including aspects of sample preparation such as protein digestion, nanoflow gradient reversed-phase chromatographic separation, LC–MS interfacing via electrospray ionization, tandem mass spectrometry of digests, protein identification via database searches, and finally peptide quantitation.


Review Proteomics research Peptide mapping Peptide profiling Nano-LC 



Alternating current


Collision-induced dissociation


Direct current


Data-dependent acquisition


Particle diameter


Electrospray ionization


Formic acid


Fast atom bombardment


False discovery rate


Heptafluorobutyric acid


High-performance liquid chromatography


Internal diameter


Isobaric tag for relative and absolute quantitation


Column length


Matrix-assisted laser desorption ionization


Multiple reaction monitoring


Tandem mass spectrometry


Mass-to-charge ratio


Plate number


Peptide-spectrum match


Quadrupole time-of-flight


Radio frequency


Reversed-phase liquid chromatography


Stable isotope labeling by amino acids in cell culture


Selected reaction monitoring


Trifluoroacetic acid




Tandem mass tag



This publication has been written as part of the Open Technology Programme with project number IWT.150467 (DEBOCS), which is financed by the Flemish Agency of Innovation and Entrepreneurship (VLAIO). JLDS and SE acknowledge the Research Foundation Flanders (FWO) for financial support (Grant nos. G 025916N and G033018N).

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.


  1. 1.
    Mallick P, Kuster B (2010) Proteomics: a pragmatic perspective. Nat Biotechnol 28:695–709CrossRefGoogle Scholar
  2. 2.
    Walsh CT (2005) Posttranslational modification of proteins: expanding nature’s inventory. Roberts and Company Publishers, Englewood, Colorado, USAGoogle Scholar
  3. 3.
    Universal Protein Resource (2018) UniProt. Accessed 4 Jul 2018
  4. 4.
    Smith JB (2001) Peptide sequencing by Edman degradation. Encyclopedia of Life Sciences. John Wiley & Sons, Chichester, UKGoogle Scholar
  5. 5.
    Barber M, Bordoli RS, Sedgwick RD, Tyler AN (1981) Fast atom bombardment of solids as an ion source in mass spectrometry. Nature 293:270–275CrossRefGoogle Scholar
  6. 6.
    Wilm M (2011) Principles of electrospray ionization. Mol Cell Proteomics 10:1–8CrossRefGoogle Scholar
  7. 7.
    Caprioli RM, Farmer TB, Gile J (1997) Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 69:4751–4760CrossRefGoogle Scholar
  8. 8.
    Wenkui Li JZ, Francis LS, Tse (2013) Handbook of LC–MS bioanalysis: best practices, experimental protocols, and regulations. Wiley, New JerseyGoogle Scholar
  9. 9.
    Chen W-Q, Obermayr P, Černigoj U, Vidič J, Panić-Janković T, Mitulović G (2017) Immobilized monolithic enzymatic reactor and its application for analysis of in-vitro fertilization media samples. Electrophoresis 38:2957–2964CrossRefGoogle Scholar
  10. 10.
    Shibue M, Mant CT, Hodges RS (2005) Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography. J Chromatogr A 1080:68–75CrossRefGoogle Scholar
  11. 11.
    Walcher W, Toll H, Ingendoh A, Huber CG (2004) Operational variables in high-performance liquid chromatography–electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths. J Chromatogr A 1053:107–117CrossRefGoogle Scholar
  12. 12.
    Hsieh EJ, Bereman MS, Durand S, Valaskovic GA, MacCoss MJ (2013) Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples. J Am Soc Mass Spectrom 24:148–153CrossRefGoogle Scholar
  13. 13.
    Yamana R, Iwasaki M, Wakabayashi M, Nakagawa M, Yamanaka S, Ishihama Y (2013) Rapid and deep profiling of human induced pluripotent stem cell proteome by one-shot NanoLC-MS/MS analysis with meter-scale monolithic silica columns. J Proteome Res 12:214–221CrossRefGoogle Scholar
  14. 14.
    Köcher T, Pichler P, Swart R, Mechtler K (2012) Analysis of protein mixtures from whole-cell extracts by single-run nanolc-ms/ms using ultralong gradients. Nat Protoc 7:882–890CrossRefGoogle Scholar
  15. 15.
    Schmidt A, Karas M, Dülcks T (2003) Effect of different solution flow rates on analyte ion signals in nano-ESI MS, or: when does ESI turn into nano-ESI? J Am Soc Mass Spectrom 14:492–500CrossRefGoogle Scholar
  16. 16.
    Chakraborty AB, Berger SJ (2005) Optimization of reversed-phase peptide liquid chromatography ultraviolet mass spectrometry analyses using an automated blending methodology. J Biomol Tech 16:325–333Google Scholar
  17. 17.
    Wang NH, Lee WL, Her GR (2011) Signal enhancement for peptide analysis in liquid chromatography–electrospray ionization mass spectrometry with trifluoroacetic acid containing mobile phase by postcolumn electrophoretic mobility control. Anal Chem 83:6163–6168CrossRefGoogle Scholar
  18. 18.
    Martin SE, Shabanowitz J, Hunt DF, Marto JA (2000) Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI fourier transform ion cyclotron resonance mass spectrometry. Anal Chem 72:4266–4274CrossRefGoogle Scholar
  19. 19.
    Reiter L, Claassen M, Schrimpf SP, Jovanovic M, Schmidt A, Buhmann JM, Hengartner MO, Aebersold R (2009) Protein identification false discovery rates for very large proteomics data sets generated by tandem mass spectrometry. Mol Cell Proteomics 8:2405–2417CrossRefGoogle Scholar
  20. 20.
    Deutsch EW, Orchard S, Binz PA et al (2017) Proteomics standards initiative: fifteen years of progress and future work. J Proteome Res 16:4288–4298CrossRefGoogle Scholar
  21. 21.
    Kohlbacher O, Reinert K, Gropl C, Lange E, Pfeifer N, Schulz-Trieglaff O, Sturm M (2007) TOPP–the OpenMS proteomics pipeline. Bioinformatics 23:e191–e197CrossRefGoogle Scholar
  22. 22.
    Köcher T, Pichler P, Swart R, Mechtler K (2011) Quality control in LC-MS/MS. Proteomics 11:1026–1030CrossRefGoogle Scholar
  23. 23.
    Mann M (2009) Comparative analysis to guide quality improvements in proteomics. Nat Methods 6:717–719CrossRefGoogle Scholar
  24. 24.
    Feist P, Hummon A (2015) Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int J Mol Sci 16:3537–3563CrossRefGoogle Scholar
  25. 25.
    Müller T, Winter D (2017) Systematic evaluation of protein reduction and alkylation reveals massive unspecific side effects by iodine-containing reagents. Mol Cell Proteomics 16:1173–1187CrossRefGoogle Scholar
  26. 26.
    Kinter M, Sherman NE (2000) Protein sequencing and identification using tandem mass spectrometry. Wiley, New JerseyCrossRefGoogle Scholar
  27. 27.
    Krenkova J, Svec F (2009) Less common applications of monoliths: IV. Recent developments in immobilized enzyme reactors for proteomics and biotechnology. J Sep Sci 32:706–718Google Scholar
  28. 28.
    Girelli AM, Mattei E (2005) Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review. J Chromatogr B 819:3–16CrossRefGoogle Scholar
  29. 29.
    Ma J, Zhang L, Liang Z, Zhang W, Zhang Y (2007) Monolith-based immobilized enzyme reactors: recent developments and applications for proteome analysis. J Sep Sci 30:3050–3059CrossRefGoogle Scholar
  30. 30.
    Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE (2009) Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol 90:10–25Google Scholar
  31. 31.
    Mirzaei H, Carrasco M (2016) Modern proteomics—sample preparation, analysis and practical applications. Springer International Publishing, ChamCrossRefGoogle Scholar
  32. 32.
    Bodzon-Kulakowska A, Bierczynska-Krzysik A, Dylag T, Drabik A, Suder P, Noga M, Jarzebinska J, Silberring J (2007) Methods for samples preparation in proteomic research. J Chromatogr B 849:1–31CrossRefGoogle Scholar
  33. 33.
    Ivanov AR, Zang L, Karger BL (2003) Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-µm-i.d. polystyrene—divinylbenzene monolithic capillary columns. Anal Chem 75:5306–5316CrossRefGoogle Scholar
  34. 34.
    Reising AE, Godinho JM, Jorgenson JW, Tallarek U (2017) Bed morphological features associated with an optimal slurry concentration for reproducible preparation of efficient capillary ultrahigh pressure liquid chromatography columns. J Chromatogr A 1504:71–82CrossRefGoogle Scholar
  35. 35.
    Godinho JM, Reising AE, Tallarek U, Jorgenson JW (2016) Implementation of high slurry concentration and sonication to pack high-efficiency, meter-long capillary ultrahigh pressure liquid chromatography columns. J Chromatogr A 1462:165–169CrossRefGoogle Scholar
  36. 36.
    Mazzeo JR, Neue UD, Kele M, Plumb RS (2005) Advancing LC performance with smaller particles and higher pressure. Anal Chem 77:460A–467ACrossRefGoogle Scholar
  37. 37.
    De Vos J, Broeckhoven K, Eeltink S (2016) Advances in ultrahigh-pressure liquid chromatography technology and system design. Anal Chem 88:262–278CrossRefGoogle Scholar
  38. 38.
    Nováková L, Vaast A, Stassen C, Broeckhoven K, De Pra M, Swart R, Desmet G, Eeltink S (2013) High-resolution peptide separations using nano-LC at ultra-high pressure. J Sep Sci 36:1192–1199CrossRefGoogle Scholar
  39. 39.
    Tolley L, Jorgenson JW, Moseley MA (2001) Very high pressure gradient LC/MS/MS. Anal Chem 73:2985–2991CrossRefGoogle Scholar
  40. 40.
    Seidler J, Adal M, Kübler D, Bossemeyer D, Lehmann WD (2009) Analysis of autophosphorylation sites in the recombinant catalytic subunit alpha of cAMP-dependent kinase by nano-UPLC–ESI–MS/MS. Anal Bioanal Chem 395:1713–1720CrossRefGoogle Scholar
  41. 41.
    Grinias KM, Godinho JM, Franklin EG, Stobaugh JT, Jorgenson JW (2016) Development of a 45kpsi ultrahigh pressure liquid chromatography instrument for gradient separations of peptides using long microcapillary columns and sub-2 µm particles. J Chromatogr A 1469:60–67CrossRefGoogle Scholar
  42. 42.
    Doneanu CE, Anderson M, Williams BJ, Lauber MA, Chakraborty A, Chen W (2015) Enhanced detection of low-abundance host cell protein impurities in high-purity monoclonal antibodies down to 1 ppm using ion mobility mass spectrometry coupled with multidimensional liquid chromatography. Anal Chem 87:10283–10291CrossRefGoogle Scholar
  43. 43.
    Davis JM, Giddings JC (1983) Statistical theory of component overlap in multicomponent chromatograms. Anal Chem 55:418–424CrossRefGoogle Scholar
  44. 44.
    Eeltink S, Wouters S, Dores-Sousa JL, Svec F (2017) Advances in organic polymer-based monolithic column technology for high-resolution liquid chromatography-mass spectrometry profiling of antibodies, intact proteins, oligonucleotides, and peptides. J Chromatogr A 1498:8–21CrossRefGoogle Scholar
  45. 45.
    Xie C, Ye M, Jiang X et al (2006) Octadecylated silica monolith capillary column with integrated nanoelectrospray ionization emitter for highly efficient proteome analysis. Mol Cell Proteomics 5:454–461CrossRefGoogle Scholar
  46. 46.
    Rozenbrand J, Van Bennekom WP (2011) Silica-based and organic monolithic capillary columns for LC: Recent trends in proteomics. J Sep Sci 34:1934–1944Google Scholar
  47. 47.
    Vaast A, Nováková L, Desmet G, de Haan B, Swart R, Eeltink S (2013) High-speed gradient separations of peptides and proteins using polymer-monolithic poly(styrene-co-divinylbenzene) capillary columns at ultra-high pressure. J Chromatogr A 1304:177–182CrossRefGoogle Scholar
  48. 48.
    Premstaller A, Oberacher H, Huber CG (2000) High-performance liquid chromatography–electrospray ionization mass spectrometry of single- and double-stranded nucleic acids using monolithic capillary columns. Anal Chem 72:4386–4393CrossRefGoogle Scholar
  49. 49.
    Eeltink S, Dolman S, Detobel F, Swart R, Ursem M, Schoenmakers PJ (2010) High-efficiency liquid chromatography–mass spectrometry separations with 50 mm, 250 mm, and 1 m long polymer-based monolithic capillary columns for the characterization of complex proteolytic digests. J Chromatogr A 1217:6610–6615CrossRefGoogle Scholar
  50. 50.
    Dolman S, Eeltink S, Vaast A, Pelzing M (2013) Investigation of carryover of peptides in nano-liquid chromatography/mass spectrometry using packed and monolithic capillary columns. J Chromatogr B 912:56–63CrossRefGoogle Scholar
  51. 51.
    Banerjee S, Mazumdar S (2012) Electrospray ionization mass spectrometry: a technique to access the information beyond the molecular weight of the analyte. Int J Anal Chem 2012:1–40CrossRefGoogle Scholar
  52. 52.
    Olsen JV, Ong S-E, Mann M (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3:608–614CrossRefGoogle Scholar
  53. 53.
    Cech NB, Enke CG (2001) Practical implications of some recent studies in electrospray ionization fundamentals. Mass Spectrom Rev 20:362–387CrossRefGoogle Scholar
  54. 54.
    Liu H, Berger SJ, Chakraborty AB, Plumb RS, Cohen SA (2002) Multidimensional chromatography coupled to electrospray ionization time-of-flight mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins. J Chromatogr B 782:267–289CrossRefGoogle Scholar
  55. 55.
    Huber CG, Premstaller A (1999) Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography–electrospray ionization mass spectrometry and capillary electrophoresis–electrospray ionization mass spectrometry of proteins: I. Liquid chromatography. J Chromatogr A 849:161–173CrossRefGoogle Scholar
  56. 56.
    Hoffmann E, Stroobant V (2007) Mass spectrometry: principles and applications. Wiley, West SussexGoogle Scholar
  57. 57.
    Paul W (1989) Nobel lecture: electromagnetic traps for charged and neutral particles. Accessed 6 Aug 2018
  58. 58.
    Hu Q, Noll RJ, Li H, Makarov A, Hardman M, Graham Cooks R (2005) The Orbitrap: a new mass spectrometer. J Mass Spectrom 40:430–443CrossRefGoogle Scholar
  59. 59.
    Syka JEP, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF (2004) Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc Natl Acad Sci USA 101:9528–9533CrossRefGoogle Scholar
  60. 60.
    Espadas G, Borras E, Chiva C, Sabido E (2017) Evaluation of different peptide fragmentation types and mass analyzers in data-dependent methods using an Orbitrap Fusion Lumos Tribrid mass spectrometer. Proteomics 17:1600416CrossRefGoogle Scholar
  61. 61.
    Hu A, Noble WS, Wolf-Yadlin A (2016) Technical advances in proteomics: new developments in data-independent acquisition. F1000Res 5:419CrossRefGoogle Scholar
  62. 62.
    Roepstorff P, Fohlman J (1984) Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom Biomed Mass Spectrom 11:601CrossRefGoogle Scholar
  63. 63.
    Ma B, Johnson R (2012) De novo sequencing and homology searching. Mol Cell Proteomics 11:O111–014902CrossRefGoogle Scholar
  64. 64.
    UniProt C (2016) UniProt: the universal protein knowledgebase. Nucleic Acids Res 45:D158–D169Google Scholar
  65. 65.
    Verheggen K, Raeder H, Berven FS, Martens L, Barsnes H, Vaudel M (2017) Anatomy and evolution of database search engines—a central component of mass spectrometry based proteomic workflows. Mass Spec Rev 2017:1–5Google Scholar
  66. 66.
    Nesvizhskii AI (2010) A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics. Proteomics 73:2092–2123CrossRefGoogle Scholar
  67. 67.
    Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389:1017–1031CrossRefGoogle Scholar
  68. 68.
    Bantscheff M, Lemeer S, Savitski MM, Kuster B (2012) Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal Bioanal Chem 404:939–965CrossRefGoogle Scholar
  69. 69.
    Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci USA 100:6940–6945CrossRefGoogle Scholar
  70. 70.
    Maccarrone G, Chen A, Filiou MD (2017) Using 15N-metabolic labeling for quantitative proteomic analyses. In: Multiplex biomarker techniques: methods and applications, vol 1546. Humana Press, New YorkGoogle Scholar
  71. 71.
    Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386CrossRefGoogle Scholar
  72. 72.
    Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904CrossRefGoogle Scholar
  73. 73.
    Wiese S, Reidegeld KA, Meyer HE, Warscheid B (2007) Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics 7:340–350CrossRefGoogle Scholar
  74. 74.
    Hsu JL, Huang SY, Chow NH, Chen SH (2003) Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem 75:6843–6852CrossRefGoogle Scholar
  75. 75.
    Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319CrossRefGoogle Scholar
  76. 76.
    Lange V, Picotti P, Domon B, Aebersold R (2008) Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 4:1–14CrossRefGoogle Scholar
  77. 77.
    Gillet LC, Navarro P, Tate S, Rost H, Selevsek N, Reiter L, Bonner R, Aebersold R (2012) Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics 11:O111-016717CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Magali Dams
    • 1
  • José Luís Dores-Sousa
    • 1
  • Robert-Jan Lamers
    • 2
  • Achim Treumann
    • 3
  • Sebastiaan Eeltink
    • 1
    Email author
  1. 1.Department of Chemical EngineeringVrije Universiteit Brussel (VUB)BrusselsBelgium
  2. 2.Abundnz B.V.WoerdenThe Netherlands
  3. 3.Newcastle University, NUPPANewcastle upon TyneUK

Personalised recommendations