Primary Structure Determination of Peptides and Enzymatically Digested Proteins Using Capillary Liquid Chromatography/Mass Spectrometry and Rapid Linked-Scan Techniques

Academic Article

Abstract

  • Primary protein sequences were determined for both peptides and enzymatically digested proteins by rapid linked-scan (B/E) liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) at the tow-picomole level (10-50 pmol). During the course of a single LC/MS/MS analysis, we demonstrated that It Is possible to generate Interpretable collison-Induced dissociation spectra of the eluting proteolytic peptides. Molecular weights of tryptic peptides were established by using 1/10 of the protein digest by operating In the capillary LC/frit-FABMS mode. Peptides exhibiting the strongest MH+ Ions were then selected for subsequent LC/MS/MS analysis (typically V, of the remaining protein dgest). Ekrtlon times for each chromatographic peak were generally >30 s. It was therefore possible to obtain a minimum of six B/E fast linked-scan spectra during the course of elution of each peptide component. Typically, B/E linked scans of the greatest Ion abundance (obtained at the chromatographic peak maximum) were averaged to enhance the slgnal/nolse ratio at these low-plcomole levels. Unit resolution was observed for product Ions below m/z 1000. Rapid linked scanning by LC/frlt-FABMS/MS provided mass assignments for product Ions within 0.2-0.3 amu of theoretical values. Side-chain fragment Ions (wn and dn) were also observed, which allowed for the differentiation of Isobarlc amino acids (e.g., leucine and isoleuclne). Examples of the application of this fast linked-scan technique to LC/MS/MS are presented for complex mixtures of unknown peptides and the tryptic digestion of phosphorylated β-casein. © 1991, American Chemical Society. All rights reserved.
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    Digital Object Identifier (doi)

    Author List

  • Smith JA; Kassel DB; Musselman BD
  • Start Page

  • 1091
  • End Page

  • 1097
  • Volume

  • 63
  • Issue

  • 11