Evaluation of Different Stationary Phases in the Separation of Inter-Cross-Linked Peptides

Abstract

Chemical crosslinking coupled with mass spectrometry (MS) is becoming a routinely and widely used technique for depicting and constructing protein structures and protein interaction networks. One major challenge for crosslinking/MS is the determination of informative low abundant intercrosslinked products, generated within a sample of high complexity. A C18 stationary phase is the conventional means for reverse-phase (RP) separation of inter-crosslinked peptides. Various RP stationary phases, which provide different selectivities and retentions, have been developed as alternatives to C18 stationary phases. In this study, two phenyl-based columns, biphenyl and fluorophenyl, were investigated and compared with a C18 phase for separating BS3 (bis(sulfosuccinimidyl)suberate) crosslinked bovine serum albumin (BSA) and myoglobin by bottom-up proteomics. Fractions from the 3 columns were collected and analyzed in a linear ion trap (LIT) mass spectrometer for improving detection of low abundant inter-crosslinked peptides. Among these three columns, the fluorophenyl column provides additional ion exchange interaction and exhibits unique retention in separating the crosslinked peptides. The fractioned data was analyzed in pLink, showing fluorophenyl column consistently obtained more inter-crosslinked peptide identifications than both C18 and biphenyl columns. For BSA crosslinked sample, the identified inter-crosslinked peptide number of fluorophenyl to C18 column are 136 to 102 in “low confident” result and 11 to 6 in “high confident” result. Fluorophenyl column could potentially be a better alternative for targeting the low stoichiometric inter-crosslinked peptides.

Synthesis, Pharmacological Evaluation, and Computational Studies of Cyclic Opioid Peptidomimetics Containing β3-Lysine

“…To confirm our observation, we subjected a mixture of these peptides to LC-MS and MS n experiments. The HPLC analysis in reversed-phase mode revealed that the isomeric peptides separate easily, using both C 18 column (Aeris Peptide) and biphenyl column (Kinetex Biphenyl), known for additional π-π interactions [ 20 ], with a nearly 0.5 min retention time difference in both cases in a 10 min gradient run from 5 to 80% acetonitrile in water ( Figure 2 and Figures S4–S6 ). Such a difference in retention time suggests altered interactions with the stationary phase, probably due to the shape of the molecules.…”

Affinity and chemical enrichment strategies for mapping low‐abundance protein modifications and protein‐interaction networks

“…Enrichment using different columns to separate cross‐linked products was also examined. Recently, a commercially available fluorophenyl column was used for the first time to enrich inter‐crosslinked peptides of BS 3 cross‐linked BSA [89]. The fact that the fluorophenyl column retained more inter‐cross‐linked peptides compared to traditional C‐18 and biphenyl columns used in this study could be attributed to greater charge‐based interactions between the negative dipole in fluorine and positively charged inter‐cross‐linked peptides [89].…”

Dual-Stage Neutral Loss Tandem Mass Spectrometric Strategy for Confident Identification of Protein Prenylation

“…Cell lysis was performed with the 1X protease inhibitor as described previously, and the protein concentration was measured with bicinchoninic acid assay using bovine serum albumin standards. Protein extractions and digestions were modified from our previous studies. , In short, 100 μg of the extracted proteins from each raw macrophage biological replicate was precipitated by the methanol/chloroform method and redissolved with 6 M guanidine hydrochloride in 50 mM ammonium bicarbonate, followed by reaction with 5 mM DTT and alkylation with 25 mM IAM in the dark. The samples were then subjected to digestion with trypsin (1:50, w/w) overnight.…”