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

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Abstract

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.

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Abbreviations

AC:

Alternating current

CID:

Collision-induced dissociation

DC:

Direct current

DDA:

Data-dependent acquisition

d p :

Particle diameter

ESI:

Electrospray ionization

FA:

Formic acid

FAB:

Fast atom bombardment

FDR:

False discovery rate

HFBA:

Heptafluorobutyric acid

HPLC:

High-performance liquid chromatography

I.D.:

Internal diameter

iTRAQ:

Isobaric tag for relative and absolute quantitation

L :

Column length

MALDI:

Matrix-assisted laser desorption ionization

MRM:

Multiple reaction monitoring

MSMS:

Tandem mass spectrometry

m/z :

Mass-to-charge ratio

N :

Plate number

PSM:

Peptide-spectrum match

QTOF:

Quadrupole time-of-flight

RF:

Radio frequency

RP-LC:

Reversed-phase liquid chromatography

SILAC:

Stable isotope labeling by amino acids in cell culture

SRM:

Selected reaction monitoring

TFA:

Trifluoroacetic acid

Th:

Thomson

TMT:

Tandem mass tag

References

  1. Mallick P, Kuster B (2010) Proteomics: a pragmatic perspective. Nat Biotechnol 28:695–709

    Article  CAS  PubMed  Google Scholar 

  2. Walsh CT (2005) Posttranslational modification of proteins: expanding nature’s inventory. Roberts and Company Publishers, Englewood, Colorado, USA

    Google Scholar 

  3. Universal Protein Resource (2018) UniProt. https://www.uniprot.org. Accessed 4 Jul 2018

  4. Smith JB (2001) Peptide sequencing by Edman degradation. Encyclopedia of Life Sciences. John Wiley & Sons, Chichester, UK

    Google Scholar 

  5. Barber M, Bordoli RS, Sedgwick RD, Tyler AN (1981) Fast atom bombardment of solids as an ion source in mass spectrometry. Nature 293:270–275

    Article  CAS  Google Scholar 

  6. Wilm M (2011) Principles of electrospray ionization. Mol Cell Proteomics 10:1–8

    Article  CAS  Google Scholar 

  7. Caprioli RM, Farmer TB, Gile J (1997) Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 69:4751–4760

    Article  CAS  PubMed  Google Scholar 

  8. Wenkui Li JZ, Francis LS, Tse (2013) Handbook of LC–MS bioanalysis: best practices, experimental protocols, and regulations. Wiley, New Jersey

    Google Scholar 

  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 38:2957–2964

    Article  CAS  PubMed  Google Scholar 

  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 1080:68–75

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 1053:107–117

    Article  CAS  PubMed  Google Scholar 

  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 24:148–153

    Article  CAS  PubMed  Google Scholar 

  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 12:214–221

    Article  CAS  PubMed  Google Scholar 

  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 7:882–890

    Article  CAS  PubMed  Google Scholar 

  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 14:492–500

    Article  CAS  PubMed  Google Scholar 

  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 16:325–333

    Google Scholar 

  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 83:6163–6168

    Article  CAS  PubMed  Google Scholar 

  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 72:4266–4274

    Article  CAS  PubMed  Google Scholar 

  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 8:2405–2417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Deutsch EW, Orchard S, Binz PA et al (2017) Proteomics standards initiative: fifteen years of progress and future work. J Proteome Res 16:4288–4298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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–e197

    Article  CAS  PubMed  Google Scholar 

  22. Köcher T, Pichler P, Swart R, Mechtler K (2011) Quality control in LC-MS/MS. Proteomics 11:1026–1030

    Article  CAS  PubMed  Google Scholar 

  23. Mann M (2009) Comparative analysis to guide quality improvements in proteomics. Nat Methods 6:717–719

    Article  CAS  PubMed  Google Scholar 

  24. Feist P, Hummon A (2015) Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int J Mol Sci 16:3537–3563

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 16:1173–1187

    Article  PubMed  PubMed Central  Google Scholar 

  26. Kinter M, Sherman NE (2000) Protein sequencing and identification using tandem mass spectrometry. Wiley, New Jersey

    Book  Google Scholar 

  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 32:706–718

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Girelli AM, Mattei E (2005) Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review. J Chromatogr B 819:3–16

    Article  CAS  Google Scholar 

  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 30:3050–3059

    Article  CAS  PubMed  Google Scholar 

  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 90:10–25

    Google Scholar 

  31. Mirzaei H, Carrasco M (2016) Modern proteomics—sample preparation, analysis and practical applications. Springer International Publishing, Cham

    Book  Google Scholar 

  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 849:1–31

    Article  CAS  Google Scholar 

  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 75:5306–5316

    Article  CAS  PubMed  Google Scholar 

  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 1504:71–82

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 1462:165–169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mazzeo JR, Neue UD, Kele M, Plumb RS (2005) Advancing LC performance with smaller particles and higher pressure. Anal Chem 77:460A–467A

    Article  CAS  Google Scholar 

  37. De Vos J, Broeckhoven K, Eeltink S (2016) Advances in ultrahigh-pressure liquid chromatography technology and system design. Anal Chem 88:262–278

    Article  CAS  PubMed  Google Scholar 

  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 36:1192–1199

    Article  CAS  PubMed  Google Scholar 

  39. Tolley L, Jorgenson JW, Moseley MA (2001) Very high pressure gradient LC/MS/MS. Anal Chem 73:2985–2991

    Article  CAS  PubMed  Google Scholar 

  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 395:1713–1720

    Article  CAS  PubMed  Google Scholar 

  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 1469:60–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 87:10283–10291

    Article  CAS  PubMed  Google Scholar 

  43. Davis JM, Giddings JC (1983) Statistical theory of component overlap in multicomponent chromatograms. Anal Chem 55:418–424

    Article  CAS  Google Scholar 

  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 1498:8–21

    Article  CAS  PubMed  Google Scholar 

  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 5:454–461

    Article  CAS  PubMed  Google Scholar 

  46. Rozenbrand J, Van Bennekom WP (2011) Silica-based and organic monolithic capillary columns for LC: Recent trends in proteomics. J Sep Sci 34:1934–1944

    CAS  PubMed  Google Scholar 

  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 1304:177–182

    Article  CAS  PubMed  Google Scholar 

  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 72:4386–4393

    Article  CAS  PubMed  Google Scholar 

  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 1217:6610–6615

    Article  CAS  PubMed  Google Scholar 

  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 912:56–63

    Article  CAS  Google Scholar 

  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 2012:1–40

    Article  CAS  Google Scholar 

  52. Olsen JV, Ong S-E, Mann M (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3:608–614

    Article  CAS  PubMed  Google Scholar 

  53. Cech NB, Enke CG (2001) Practical implications of some recent studies in electrospray ionization fundamentals. Mass Spectrom Rev 20:362–387

    Article  CAS  PubMed  Google Scholar 

  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 782:267–289

    Article  CAS  Google Scholar 

  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 849:161–173

    Article  CAS  PubMed  Google Scholar 

  56. Hoffmann E, Stroobant V (2007) Mass spectrometry: principles and applications. Wiley, West Sussex

    Google Scholar 

  57. Paul W (1989) Nobel lecture: electromagnetic traps for charged and neutral particles. http://www.nobelprize.org. Accessed 6 Aug 2018

  58. Hu Q, Noll RJ, Li H, Makarov A, Hardman M, Graham Cooks R (2005) The Orbitrap: a new mass spectrometer. J Mass Spectrom 40:430–443

    Article  CAS  PubMed  Google Scholar 

  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 101:9528–9533

    Article  CAS  PubMed  Google Scholar 

  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 17:1600416

    Article  CAS  Google Scholar 

  61. Hu A, Noble WS, Wolf-Yadlin A (2016) Technical advances in proteomics: new developments in data-independent acquisition. F1000Res 5:419

    Article  Google Scholar 

  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 11:601

    Article  CAS  PubMed  Google Scholar 

  63. Ma B, Johnson R (2012) De novo sequencing and homology searching. Mol Cell Proteomics 11:O111–014902

    Article  CAS  PubMed  Google Scholar 

  64. UniProt C (2016) UniProt: the universal protein knowledgebase. Nucleic Acids Res 45:D158–D169

    Google Scholar 

  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 2017:1–5

    Google Scholar 

  66. Nesvizhskii AI (2010) A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics. Proteomics 73:2092–2123

    Article  CAS  PubMed  Google Scholar 

  67. Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389:1017–1031

    Article  CAS  PubMed  Google Scholar 

  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 404:939–965

    Article  CAS  PubMed  Google Scholar 

  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 100:6940–6945

    Article  CAS  PubMed  Google Scholar 

  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 York

    Google Scholar 

  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 1:376–386

    Article  CAS  Google Scholar 

  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 75:1895–1904

    Article  CAS  PubMed  Google Scholar 

  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 7:340–350

    Article  PubMed  Google Scholar 

  74. Hsu JL, Huang SY, Chow NH, Chen SH (2003) Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem 75:6843–6852

    Article  CAS  PubMed  Google Scholar 

  75. Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319

    Article  CAS  PubMed  Google Scholar 

  76. Lange V, Picotti P, Domon B, Aebersold R (2008) Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 4:1–14

    Article  Google Scholar 

  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:O111-016717

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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).

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Authors and Affiliations

  1. Department of Chemical Engineering, Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050, Brussels, Belgium

    Magali Dams, José Luís Dores-Sousa & Sebastiaan Eeltink

  2. Abundnz B.V., Woerden, The Netherlands

    Robert-Jan Lamers

  3. Newcastle University, NUPPA, Newcastle upon Tyne, UK

    Achim Treumann

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  1. Magali Dams
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  2. José Luís Dores-Sousa
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  3. Robert-Jan Lamers
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  4. Achim Treumann
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  5. Sebastiaan Eeltink
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Correspondence to Sebastiaan Eeltink.

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Published in Chromatographia’s 50th Anniversary Commemorative Issue.

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Dams, M., Dores-Sousa, J.L., Lamers, RJ. et al. High-Resolution Nano-Liquid Chromatography with Tandem Mass Spectrometric Detection for the Bottom-Up Analysis of Complex Proteomic Samples. Chromatographia 82, 101–110 (2019). https://doi.org/10.1007/s10337-018-3647-5

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