Near edge X-ray absorption mass spectrometry of gas phase proteins: the influence of protein size

Abstract

Multiply protonated peptides and proteins in the gas phase can respond to near edge X-ray absorption in three different ways: (i) non dissociative ionization and ionization accompanied by loss of small neutrals, both known to dominate for proteins with masses in the 10 kDa range. (ii) Formation of immonium ions, dominating for peptides in the 1 kDa range. (iii) Backbone scission leading to sequence ions which is typically weaker and has mainly been observed for peptides in the 1 kDa range. We have studied carbon 1s photoexcitation and photoionization for a series of peptides and proteins with masses covering the range from 0.5 kDa to more than 10 kDa. The gas phase protonated molecules were trapped in a radiofrequency ion trap and exposed to synchrotron radiation. Time of flight mass spectrometry was employed for investigation of the photoionization and photofragmentation processes. A smooth transition from the photofragmentation regime to the non-dissociative photoionization regime is observed. Mass spectra are most complex in the few kDa regime, where non-dissociative ionization, backbone scission and immonium ion formation coexist. The observed correlation between protein size and fragmentation, i.e. radiation damage, is of relevance for soft X-ray microscopy.

Single-photon absorption of isolated collagen mimetic peptides and triple-helix models in the VUV-X energy range

“…We attribute the main ones to internal fragments formed by at least two backbone cleavages (see figure 3a). This is in line with our previously reported mass spectra of peptides after X-ray single photon absorption: we showed that peptides of similar size or smaller undergo extensive fragmentation into low-mass ions due to one or two bond cleavages 12,20 .…”

“…We recently reported such a size effect for a range of peptides and proteins 12 . For soft X-ray photon energies of 150 and 288 eV, we observe the appearance of peaks in the low m/z range which might be due to a high amount of internal energy deposited in [((PPG) 10 ) 3 +7H] 7+ .…”

“…Previous investigations on biologically-relevant molecular systems in the gas phase by means of VUV to soft X-ray synchrotron radiation have revealed that proteins, oligonucleotides and their building blocks (amino acids, peptides, nucleobases…) can be photoionized by single photon absorption via electron removal from valence (VUV) or atomic (X-ray) orbitals 11 . The accompanying electronic excitation can induce fragmentation if the molecular heat capacity is small enough that sufficient internal temperatures for thermal fragmentation are reached 12 . Reports on photoionization of non-covalent complexes of biomolecules are scarce [13][14][15] , mainly focused on the binding site of a ligand to a protein, and have not yet covered the X-ray range.…”

Soft X‐ray Spectroscopy as a Probe for Gas‐Phase Protein Structure: Electron Impact Ionization from Within

“…For melittin at room temperature, these channels are known to coexist 16. At T ≈10 K, in contrast, our data show that non‐dissociative single and double ionization become the strongest channels.…”

“…[15] For melittin at room temperature, these channels are knownt ocoexist. [16] At T % 10 K, in contrast, our data show that non-dissociatives ingle and double ionization become the strongest channels. Figure 1d isplays the experimental data for non-dissociative single ionization (NDSI) of [melittin + qH] q + (q = 2-4).…”