Multidimensional nano-HPLC for analysis of protein complexes.
The analysis of macromolecular protein complexes is an important factor in understanding most cellular processes, e.g., protein transport into cell organells, signal transduction via biological membranes, apoptosis, energy metabolism, directed motion of cells, and cell division. These complexes are not only built of various numbers of different proteins but also of prosthetic groups and RNA molecules. To understand the role each protein plays in a complex, a complete analysis of all protein compounds is necessary. Therefore, several separation steps have to be coupled to mass spectrometry to identify the proteins. In this work, we describe the application of multidimensional liquid chromatography, SCX-RP-LC as well as SAX-RP-LC, coupled to electrospray ion trap mass spectrometry. Tryptic digested ribosomes were separated by ion exchange chromatography manually collected and prepared for reversed phase chromatography to analyze the peptides via nano-ESI mass spectrometry. The total numbers of identified proteins are compared in consideration of the separation method (SCX-RP versus SAX-RP).
Wagner
Yvonne
Y
Rudolf Virchow-Zentrum, Julius Maximilians University Wuerzberg, Wuerzburg, Germany.
Sickmann
Albert
A
Meyer
Helmut E
HE
Daum
Günther
G
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods*; D016366:Open Reading Frames / Q000235:genetics; D015203:Reproducibility of Results; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
D005656:Fungal Proteins; D046911:Macromolecular Substances; D014357:Trypsin