Premium
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
Author(s) -
Wu ShiawLin,
Jardine Ian,
Hancock William S.,
Karger Barry L.
Publication year - 2004
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1609
Subject(s) - chemistry , fourier transform ion cyclotron resonance , mass spectrometry , quadrupole ion trap , top down proteomics , chromatography , ion trap , lysis , ion cyclotron resonance , mass spectrum , protein mass spectrometry , ion , analytical chemistry (journal) , tandem mass spectrometry , biochemistry , organic chemistry , cyclotron
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 purified from inclusion bodies. On‐line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on‐line LC/MS top‐down method. Copyright © 2004 John Wiley & Sons, Ltd.