Direct Identification of Intact Proteins Using a Low-Resolution Mass Spectrometer with CIDⁿ/ETnoD

Over the past decades, proteomics has become increasingly important and a heavily discussed topic. The identification of intact proteins remains a major focus in this field. While most intact proteins are analyzed using high-resolution mass spectrometry, identifying them through low-resolution mass spectrometry continues to pose challenges. In our study, we investigated the capability of identifying various intact proteins using collision-induced dissociation (CID) and electron transfer without dissociation (ETnoD). Using myoglobin as our test protein, stable product ions were generated with CID, and the identities of the product ions were identified with ETnoD. ETnoD uses a short activation time (AcT, 5 ms) to create sequential charge-reduced precursor ion (CRI). The charges of the fragments and their sequences were determined with corresponding CRI. The product ions can be selected for subsequent CID (termed CIDⁿ) combined with ETnoD for further sequence identification and validation. We refer to this method as CIDⁿ/ETnoD. The use of a multistage CID activation (CIDⁿ) and ETnoD protocol has been applied to several intact proteins to obtain multiple sequence identifications.