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Proteins can be separated first by pI during isoelectric focusing followed by molecular weight separation on a polyacrylamide gel. After in-gel tryptic digestion, the peptide products are introduced into to the mass spectrometer for LC-MS analysis. The spectra containing the peptide fingerprints can then be searched using Mascot. One advantage to using peptide mass fingerprinting for protein identification is that the molecular weight and pI information can be incorporated into Mascot searches to increase the confidence of the results. HRT mass spectrometry analysis of protein standards digested with trypsin introduced using static nanospray shows that protein mixtures can be reliably identified using the Mascot search algorithm provided that no more than two proteins are present in the mixture. HRT spectra from these empirical experiments showed good resolution (50,000) and high mass accuracy (<1 ppm). It should also be noted that the sensitivity can be increased (> 3-fold) using velocity modulation and the resolution increased to (<70,000) using zoom mode while still preserving mass accuracy. In-silco analysis of 500 proteins from the S. cerevisiae Swiss-Prot database reveals, that the likelihood of having a protein mixture of two a more proteins is 5.98% if the pI resolution equals 1 pH unit and MW resolution equals 500 Da; however, a more optimal separation of the proteins where the pI resolution equals 0.05 pH unit and MW resolution equals 100 Da shows that protein mixtures of two or more proteins occur at a frequency of less than 1%. By achieving optimal protein separation, digested proteins can be directly infused into the mass spectrometer using static nanospray. Without further LC-based separation, proteins can be identified more expediently without sacrificing the ability to reliably identify them using peptide mass fingerprinting.