A method for identifying one or more microbes, comprising: a. providing a sample containing one or more microbes; b. disrupting the one or more microbes present in the sample to provide a fluid extract; c. separating a soluble protein fraction from an insoluble protein fraction present in the fluid extract and preparing a solution containing intact soluble proteins from the one or more microbes; d. ionizing the intact soluble proteins by means of an ion source of a mass spectrometer to form ionized proteins; e. analyzing the ionized proteins with a mass analyzer of the mass spectrometer, wherein analyzing comprises: i. in a first mass spectrometry step, acquiring one or more mass spectra representative of one or more of the ionized intact proteins; ii. determining molecular weights for the ionized intact proteins from the one or more mass spectra; iii. using the determined molecular weights of the ionized intact proteins to search a first database containing highly accurate molecular weights of known microbial proteins and matching the determined molecular weights with the highly accurate molecular weights to select a reduced number of possible microbial identification candidates; iv. in a second mass spectrometry step, selecting one or more precursor ions of the proteins from the one or more ionized proteins of the sample and fragmenting the precursor ions by fragmentation means to produce a plurality of product ions; v. using mass-to-charge ratios of the plurality of product ions to search a second database containing molecular weights of known microbial proteins and at least one of product ion m/z values, amino acid sequence, or post-translational modification information of said known microbial proteins, wherein the subset of candidate microbes from the first database is used to limit a search of the second database; and f. automatically analyzing a sufficient number of proteins to identify at least one of the one or more microbes in said sample; wherein steps e(i) to e(iii) are performed in real time, and wherein steps (b) to (f) are performed in less than 10 minutes, preferably in less than 5 minutes, more preferably within about 1 minute or less. a. providing a sample containing one or more microbes; b. disrupting the one or more microbes present in the sample to provide a fluid extract; c. separating a soluble protein fraction from an insoluble protein fraction present in the fluid extract and preparing a solution containing intact soluble proteins from the one or more microbes; d. ionizing the intact soluble proteins by means of an ion source of a mass spectrometer to form ionized proteins; e. analyzing the ionized proteins with a mass analyzer of the mass spectrometer, wherein analyzing comprises: i. in a first mass spectrometry step, acquiring one or more mass spectra representative of one or more of the ionized intact proteins; ii. determining molecular weights for the ionized intact proteins from the one or more mass spectra; iii. using the determined molecular weights of the ionized intact proteins to search a first database containing highly accurate molecular weights of known microbial proteins and matching the determined molecular weights with the highly accurate molecular weights to select a reduced number of possible microbial identification candidates; iv. in a second mass spectrometry step, selecting one or more precursor ions of the proteins from the one or more ionized proteins of the sample and fragmenting the precursor ions by fragmentation means to produce a plurality of product ions; v. using mass-to-charge ratios of the plurality of product ions to search a second database containing molecular weights of known microbial proteins and at least one of product ion m/z values, amino acid sequence, or post-translational modification information of said known microbial proteins, wherein the subset of candidate microbes from the first database is used to limit a search of the second database; and i. in a first mass spectrometry step, acquiring one or more mass spectra representative of one or more of the ionized intact proteins; ii. determining molecular weights for the ionized intact proteins from the one or more mass spectra; iii. using the determined molecular weights of the ionized intact proteins to search a first database containing highly accurate molecular weights of known microbial proteins and matching the determined molecular weights with the highly accurate molecular weights to select a reduced number of possible microbial identification candidates; iv. in a second mass spectrometry step, selecting one or more precursor ions of the proteins from the one or more ionized proteins of the sample and fragmenting the precursor ions by fragmentation means to produce a plurality of product ions; v. using mass-to-charge ratios of the plurality of product ions to search a second database containing molecular weights of known microbial proteins and at least one of product ion m/z values, amino acid sequence, or post-translational modification information of said known microbial proteins, wherein the subset of candidate microbes from the first database is used to limit a search of the second database; and f. automatically analyzing a sufficient number of proteins to identify at least one of the one or more microbes in said sample;