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Fully Automated Nano-Electrospray Coupled with a Finnigan LCQ Deca XP Plus for Sub-fmol Proteomic Analysis

ter which accepts only peptides with Xcorr values of 1.5, 2.0, and 2.5 and higher for singly, doubly, or triply charged precursor ions.

Results and Discussion
The tryptic digest of horse myoglobin was analyzed by nanospray liquid chromatography as described in the Experimental section. The LC/MS system was run under standard conditions one would use for typical protein identification in a complex mixture of proteins and not optimized for the detection of horse myoglobin digest. The mass range for full mass spectrum was 400 Da to 1500 Da and each full mass spectrum was followed by three MS/MS scans of the most intense ions. Dynamic Exclusion was enabled. Horse myoglobin could be identified in amounts as low as 500 amol. Figure 3 shows the BioWorks results and Figure 4 shows one of two tandem mass spectra obtained for this low concentration. Both tandem mass spectra contained sufficient fragment ions for positive identification by the SEQUEST algorithm. The results were confirmed several times after rigorous washing of the LC (peptide trapPicoFrit column) system.

The Finnigan Surveyor HPLC system, used with an optimized flow splitting system, generated very reproducible chromatograms at flow rates of 100 nL/min. Two different amounts (250 fmol and 500 fmol) of a tryptic myoglobin digest were analyzed three time each and the elution time of each peptide differs by less than 10 seconds from run to run, which is less than 0.3% of the elution time (Figure 5).

Overall system reproducibility was further evaluated by analyzing a mixture of all five standard protein digests at various concentrations ranging from 10 fmols to 6 pmols. Forty replicate analyses were performed. All five proteins could be identified in each of the 40 analyses, using BioWorks software. Peptide coverage for each protein was very similar between all runs, demonstrating excellent reproducibility of LC/MS with t
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Mouse monoclonal [3G131] to Desmoglein 1 ( Abpromise for all tested applications). entrezGeneID: 1828 SwissProtID: Q02413...
TCP-1 alpha (H-110)...
Synphilin 1 Immunogen: Synthetic peptide corresponding to residues S(829) L E L N G E K D K D K G R T L Q R T(847) of human Synphilin 1. Storage: -20 C, Avoid Freeze/Thaw Cycles...
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