Multiplexing the Identification of Microorganisms via Tandem Mass Tag Labeling Augmented by Interference Removal through a Novel Modification of the Expectation Maximization Algorithm.

Having fast, accurate, and broad spectrum methods for the identification of microorganisms is of paramount importance to public health, research, and safety. Bottom-up mass spectrometer-based proteomics has emerged as an effective tool for the accurate identification of microorganisms from microbial isolates. However, one major hurdle that limits the deployment of this tool for routine clinical diagnosis, and other areas of research such as culturomics, is the instrument time required for the mass spectrometer to analyze a single sample, which can take ∼1 h per sample, when using mass spectrometers that are presently used in most institutes. To address this issue, in this study, we employed, for the first time, tandem mass tags (TMTs) in multiplex identifications of microorganisms from multiple TMT-labeled samples in one MS/MS experiment. A difficulty encountered when using TMT labeling is the presence of interference in the measured intensities of TMT reporter ions. To correct for interference, we employed in the proposed method a modified version of the expectation maximization (EM) algorithm that redistributes the signal from ion interference back to the correct TMT-labeled samples. We have evaluated the sensitivity and specificity of the proposed method using 94 MS/MS experiments (covering a broad range of protein concentration ratios across TMT-labeled channels and experimental parameters), containing a total of 1931 true positive TMT-labeled channels and 317 true negative TMT-labeled channels. The results of the evaluation show that the proposed method has an identification sensitivity of 93-97% and a specificity of 100% at the species level. Furthermore, as a proof of concept, using an in-house-generated data set composed of some of the most common urinary tract pathogens, we demonstrated that by using the proposed method the mass spectrometer time required per sample, using a 1 h LC-MS/MS run, can be reduced to 10 and 6 min when samples are labeled with TMT-6 and TMT-10, respectively. The proposed method can also be used along with Orbitrap mass spectrometers that have faster MS/MS acquisition rates, like the recently released Orbitrap Astral mass spectrometer, to further reduce the mass spectrometer time required per sample.