We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer.The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-coA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.
We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N, N, N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manud or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.
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<p> We present an acoustic ejection mass spectrometry (AEMS) setup for ESI-MS based sample </p><p> injection at a sampling rate faster than current ESI and MALDI techniques. A modified acoustic </p><p> droplet ejection system was combined with an open port interface and a modified ESI source. To </p><p> simulate applications of drug metabolism and pharmacokinetics analysis and high-throughput </p><p> screening campaigns, two stress tests were performed regarding ion suppression and system </p><p> endurance in combination with minor sample preparation. Maximum sampling rate was 6 Hz for </p><p> dextromethorphan and d3-dextrorphan (each 100 nM) for 1152 injections in 63 s at FWHM of </p><p> 105 ms and %RSD of 7.7%/7.5% without internal standard correction. Enzyme assay buffer and </p><p> crude dog plasma caused signal suppression of 51%/73% at %RSD of 5.7%/6.7% (n = 120) and </p><p> stable OPI performance during 1100 injections. An endurance buffer revealed minor OPI pollution </p><p> and constant signals for >25.000 injections (%RSD = 8.5%, n = 10,557). </p></div>
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