On the Origin of “Picket Fence” Peaks: Spectral Complexity, Mass Defect Uniformity, and Mass-to-Charge Ratio Alignment in Tandem Mass Spectra of Intact Proteins

Abstract Spectra from tandem mass spectrometry (MS/MS) of intact proteins are often highly complex, containing numerous closely spaced peaks arising from fragment ions with similar m/z values. This high level of spectral density continues to challenge even state-of-the-art high-resolution mass spectrometers, particularly as protein size increases. Here, we show that this complexity can produce a misleading spectral interference pattern when dissociating proteins whose fragment ions exhibit a sufficiently linear relationship between fragment mass and mass defect. In such cases, isotopic m/z peaks from multiple fragment ions constructively overlap to produce a series of predominantly ∼1 m/z-spaced peaks, which we refer to as a “picket-fence” pattern. These peaks are easily misinterpreted as signals from much lower mass fragment ions (“peak at every mass” background) or distort expected isotopic peak envelopes. More fundamentally, these picket fence peaks can be understood as the collective manifestation of a high spectral density of signals arising from both sequence ions and the aggregate contribution of numerous individually low-abundance internal fragment ions. Recognizing and accounting for this often-observed phenomenon is essential for accurate fragment-ion assignment and interpretation of intact protein MS/MS spectra.