Analysis of barley chromosomal proteome: a multiapproach exploration of flow cytometry-sorted mitotic chromosomes

Proteins contribute to the structure of chromatin and regulate its functions. The basic building block of chromatin in eukaryotes is the nucleosome containing histones. Structural maintenance of chromosomes complexes perform the assembly of genomic DNA into the higher organizational structures of interphase chromatin as well as the compact arrangement of dividing chromosomes. Here, proteins were extracted from flow cytometry-sorted barley mitotic chromosomes after a nuclease treatment to remove DNA. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system prior to liquid chromatography coupled to tandem mass spectrometry. Nuclear and chromosomal proteins (almost 900 identifications) were then classified based on a combination of software prediction results, available database localization information, sequence homology, and domain representation. The degree of enrichment of the chromosome samples by chromosomal proteins was evaluated by comparing the results with controls (depleted flow cytometric fraction and initial cell lyzate). A biological context evaluation indicated the presence of several groups of abundant proteins including histone variants, topoisomerase 2, PARP2, Condensin complex subunits, and many proteins with chromatin-related functions. Interestingly, nucleolar proteins were found as wells as proteins documenting processes related to replication, transcription and DNA repair in barley mitotic chromosomes.