New reagents for enhanced liquid chromatographic separation and charging of intact protein ions for electrospray ionization mass spectrometry.
Electrospray ionization produces multiply charged ions, thereby lowering the mass-to-charge ratio for peptides and small proteins to a range readily accessed by quadrupole ion trap, orbitrap, and ion cyclotron resonance (ICR) mass analyzers (m/z = 400-2000). For Fourier transform mass analyzers (orbitrap and ICR), higher charge also improves signal-to-noise ratio, mass resolution, and mass accuracy. Addition of m-nitrobenzyl alcohol (m-NBA) or sulfolane has previously been shown to increase the charge states of proteins. Moreover, polar aprotic dimethylformamide (DMF) improves chromatographic separation of proteolytic peptides for mass analysis of solution-phase protein hydrogen/deuterium exchange for improved (78-96%) sequence coverage. Here, we show that addition of each of the various modifiers (DMF, thiodiglycol, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone) can significantly increase the charge states of proteins up to 78 kDa. Moreover, incorporation of the same modifiers into reversed-phase liquid chromatography solvents improves sensitivity, charging, and chromatographic resolution for intact proteins.
Valeja
Santosh G
SG
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, USA.
Tipton
Jeremiah D
JD
Emmett
Mark R
MR
Marshall
Alan G
AG
D000081:Acetamides / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004126:Dimethylformamide / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011760:Pyrrolidinones / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013876:Thiophenes / Q000737:chemistry
D000081:Acetamides; D010455:Peptides; D011506:Proteins; D011760:Pyrrolidinones; D013438:Sulfhydryl Compounds; D013876:Thiophenes; D004126:Dimethylformamide; C047519:2,2'-thiodiethanol; C013959:dimethylacetamide; C038678:N-methylpyrrolidone; C013693:sulfolane; D004121:Dimethyl Sulfoxide