A simple and inexpensive approach to interfacing high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry.
The ability to obtain the accurate mass of a protein in a complex sample mixture aids in determining its correct in vivo form. This is important when identifying post-translationally modified proteins, protein variants or isoforms. The central technique used to separate proteins, 2-dimensional gel electrophoresis offers excellent separation capabilities but does not provide adequate mass accuracy. In this study, an alternative method, liquid chromatography (LC) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-MS (LC-MALDI) is described. LC-MALDI-MS was used to separate and determine the mass of proteins and peptides in a complex biological sample (i.e., human pituitary gland homogenate). Peptides and proteins were first separated by capillary chromatography and the eluent mixed post-column with sinapinic acid matrix. The flow was then deposited directly onto a standard MALDI target via a capillary nebulizer. In addition to offering high mass accuracy, this method can be applied to peptide and protein quantification.
Fung
Kim Y C
KY
Biochemical Mass Spectrometry Facility, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Askovic
Srdjan
S
Basile
Franco
F
Duncan
Mark W
MW
D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000191:economics* / Q000379:methods*; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D010902:Pituitary Gland / Q000378:metabolism; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000191:economics* / Q000379:methods*; D013223:Statistics as Topic
D010455:Peptides; D020033:Protein Isoforms