A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer

Rapid Communications in Mass Spectrometry : RCM
Shiaw-Lin WuBarry Karger

Abstract

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH pur...Continue Reading

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Citations

Oct 29, 2005·Journal of the American Society for Mass Spectrometry·Vlad ZabrouskovNeil L Kelleher
Mar 17, 2006·Journal of the American Society for Mass Spectrometry·Facundo M FernandezJulia Laskin
Jul 4, 2006·NeuroRx : the Journal of the American Society for Experimental NeuroTherapeutics·Thomas J MontineJing Zhang
Oct 3, 2012·Molecular & Cellular Proteomics : MCP·Chenxi JiaLingjun Li
Jul 12, 2014·Annual Review of Analytical Chemistry·Zhaorui ZhangLjiljana Paša-Tolić
Jul 8, 2005·Expert Review of Proteomics·Julia Tait LathropDavid J Hammond
Feb 16, 2006·Molecular & Cellular Proteomics : MCP·Boris MacekMatthias Mann

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