Application of the Crosslink Analysis Strategy to the Red Cell Membrane

While the CX-MS analysis strategy described in this dissertation is capable to

achieving high-confidence crosslink identifications with low FDR regardless of protein

complex sizes, without additional experimental steps, the depth-of-analysis declines

rapidly as the protein complex size increases. This is due to two limiting factors of the

mass spectrometer instruments. First, only up to a certain amount of peptide content can

be analyzed in each LC-MS/MS run (e.g. about 1µg when 75µm ID columns are used)

without inducing distortions in the chromatographic profile. Hence, as protein complex

size increases, the number of molecules of a specific crosslinked peptide that can be

analyzed in a single run decreases, resulting in reduced crosslinked peptide signal

intensities. Second, high-resolution mass analyzers such as the Orbitrap require minimum

peptide signal intensities in order to generate good-quality MS/MS spectra. In our case,

the signal intensity threshold was set at 50,000 ion counts. Therefore, if a protein

complex size increases by 10-fold, the femtomoles of the crosslinked sample that can be

injected into the mass spectrometer will drop by 10-fold and the signal intensities of

many crosslinked peptides will fall below the detection threshold. A straightforward

109

splitting it into largely non-overlapping portions in terms of peptide content. This method

allows larger amounts of crosslinked peptides to be analyzed at the penalty of an

increased number of LC-MS/MS runs. It is important to note here that the fractionation

technique required here is fundamentally different from those described in (68-69), as the

main purpose here is to distribute crosslinked peptides across fractions as uniformly as

possible. Preliminary fractionation efforts using red cell membranes crosslinked with

EDC confirm that this strategy can greatly improve the depth of analysis for very

110

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