Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases.
A method is described for improving the sensitivity of peptide mapping with electrospray liquid chromatography--mass spectrometry using trifluoroacetic acid (TFA) containing HPLC mobile phases. The signal suppressing effects of TFA are shown to be due to the combined effect of ion-pairing and surface tension modifications. The post-column addition of a propionic acid-2-propanol (75:25, v/v) in a 1:2 proportion with the HPLC mobile phase counteracts the deleterious effects of TFA resulting in 10-100 x improvement of the signal-to-noise ratio. The system described introduces total HPLC flow (plus additive) directly into the electrospray source without splitting. Using 2.1 mm I.D. HPLC columns, minimum detectable quantities are below 40 pmol total protein. As examples, separations of proteolytic enzyme digests of several proteins are shown using standard HPLC conditions, comparing results with and without the addition of propionic acid. The application of the technique is shown in more depth in the identification of oxidative modification sites in glutamine synthetase. In this application, the enhanced sensitivity allowed location of a modified residue by comparison endoproteinase Lys C digest of native and oxidized forms of the protein without extensive sample preparation or concentration. A third application demonstrates the identification of glycosylation sites in an endoproteinase Arg C digest of single-chain plasminogen activator through the use of in-source collisionally induced dissociation.
Apffel
A
A
Bay Analytical Operation, Hewlett-Packard Co., Palo Alto, CA 94304, USA.
Fischer
S
S
Goldberg
G
G
Goodley
P C
PC
Kuhlmann
F E
FE
D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D010450:Endopeptidases / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry; D006031:Glycosylation; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data; D009113:Muramidase / Q000737:chemistry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010960:Plasminogen Activators / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D012997:Solvents; D014269:Trifluoroacetic Acid
D007202:Indicators and Reagents; D010446:Peptide Fragments; D012997:Solvents; D014269:Trifluoroacetic Acid; D009113:Muramidase; D010450:Endopeptidases; D010960:Plasminogen Activators; D005974:Glutamate-Ammonia Ligase