Systems and methods described herein can provide for top down mass spectrometric analysis of proteins or peptides in a sample using ExD, in some aspects via direct infusion of the sample to the ion source without on-line LC separation, while deconvoluting the ambiguity in the ExD spectra generated by impure samples. For example, methods and systems in accordance with various aspects of the present teachings can utilize patterns in charge-reduced species following ExD to correlate the ExD fragments with their precursor ions in order to more confidently identify the precursor ion from which the detected product ions originated.

An ion sequestering apparatus and methods or systems using one or more auxiliary electrodes in an ion reaction instrument having RF electrodes adapted to guide positively-charged precursor ions along a first axis, and an electron source for introduction of an electron beam along a second axis transverse to the first axis such that electron activated dissociation of the precursor ions into reaction products can occur, the auxiliary electrode configured to apply a supplemental AC signal to permit selective extraction of reaction products while sequestering precursor ions along the second central axis. For example, the supplemental AC signal can comprises an notched white noise signal with a notch that suppresses frequencies at which the precursor ions (and/or charge reduced species that have the same molecular mass but have a different charge state) would otherwise be excited.

A method of performing negative electron activation dissociation (negative EAD) in mass spectrometry includes introducing a plurality of negatively charged analyte ions into an ion trap positioned in a chamber and trapping said negatively charged analyte ions in a reaction region of said ion trap, introducing a buffer gas into the chamber, using an electron source positioned in the chamber and external to the ion trap to generate electrons, and accelerating the electrons to form an electron beam and introducing the electron beam into the ion trap such that the accelerated electrons are capable of ionizing at least a portion of molecules of the buffer gas to generate a plurality of positively charged ions. The accelerated electrons interact with at least a portion of the analyte ions trapped in said reaction region to cause negative EAD thereof, thereby generating a plurality of fragment product ions.

A separation time of an isomer of one or more isomers of a sialylated glycopeptide of a sample is calculated from a peak of a precursor XIC. Product ion intensities of the first group are summed at the separation time producing a first sum and product ion intensities of the second group are summed at the separation time producing a second sum using XICs of the first and second groups. A ratio of the first sum to the second sum is calculated. The ratio at the separation time is compared to predetermined ratio ranges that each corresponds to a combination of a selection from a set of the first linkage and the second linkage taken one or more times. One or more linkages of the sialic acid to the glycan of the isomer are identified from a combination found to match the ratio in the comparison.

A method for mass spectrometric analysis of a peptide having at least one fragile moiety includes using electrospray ionization to generate a negatively charged ion of said peptide, trapping and cooling the negatively charged peptide ion in a radiofrequency (RF) ion trap containing a cooling buffer gas, and exposing said cooled, trapped peptide ion to an electron beam so as to cause negative electron activated dissociation (negative EAD) of the negatively charged peptide ion to generate a plurality of fragment ions.