Analysis of tartary buckwheat ( Fagopyrum tataricum ) seed proteome using offline two‐dimensional liquid chromatography and tandem mass spectrometry

Abstract

The whole seed of tartary buckwheat (Fagopyrum tataricum) is considered as a healthy and functional food, which is rich in kinds of flavonoids and with potential antioxidant effect. An in‐depth analysis of tartary buckwheat seed (TBS) proteome was performed using a shotgun proteomics strategy. Total protein of TBS was extracted and digested, then the peptides were separated by offline two‐dimensional liquid chromatography and identified by tandem mass spectrometry. Total of 3,363 high‐confidence proteins were identified from 13,730 matched peptides, in which, 2,499 proteins were annotated by the Gene Ontology (GO) analysis with 1,720 involved in “biological process,” 2,241 in “molecular function,” and 693 in “cellular components.” Based on the GO functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment results, buckwheat seed proteins were mostly enriched in metabolism of nucleic acid, respiration and energy metabolism, as well as synthesis and metabolism of protein. Practical applications This study characterized the tartary buckwheat seed proteome on a scale of 3,000+ proteins and provide important information and clues for future research, especially in the mechanism of seed germination, nutrient composition changes, and metabolite production seed germination and material metabolism.

A puzzle piece of protein N-glycosylation in chicken egg: N-glycoproteome of chicken egg vitelline membrane

“…The sample was then diluted to a urea concentration of less than 2 mol/L by adding NH 4 HCO 3 (100 mmol/L). Digestion was performed by adding trypsin at a mass ratio of 1:50 (trypsin:protein) overnight, followed by a second digestion at a mass ratio of 1:50 (trypsin:protein) for 4 h [ 14 , 15 ].…”

In‐depth mapping of the proteome of Tibetan pig tenderloin ( longissimus dorsi ) using offline high‐pH reversed‐phase fractionation and LC‐MS/MS

“…Afterward, the samples were diluted by adding 100 mM triethylamine borane (TEAB) to achieve a urea concentration of less than 2 mol/L. Finally, samples were digested using trypsin (Sigma‐Aldrich) at a 1:50 trypsin‐to‐protein mass ratio for the first digestion overnight and at a 1:100 trypsin‐to‐protein mass ratio for the second 4 hr digestion (Wang, Xiao, Geng, et al, ; Wang, Xiao, Liu, et al, ).…”

“…The peptides were loaded onto a Reprosil‐Pur C18 reverse‐phase analytical column (1.9 μm particles, inner diameter 75 µm, 15 cm length) and separated with a gradient of buffer A (2% acetonitrile with 0.1% formic acid) and buffer B (90% acetonitrile with 0.1% formic acid) at a flow rate of 300 nl/min. Buffer B increased from 9% to 25% in 24 min and increased from 25% to 36% in 8 min, then increased to 80% in 4 min, where it was held for 4 min before decreasing back to 9% B (Geng et al, ; Wang, Xiao, Liu, et al, ).…”

“…Subsequently, the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/pathway.html) database was used to annotate the protein pathway. First, the KEGG online service tool KAAS was used to annotated protein's KEGG database description; then, the annotation results were mapped on the KEGG pathway database using the KEGG online service tool KEGG mapper (Geng et al, ; Wang, Xiao, Liu, et al, ).…”

“…In recent years, two‐dimensional liquid chromatography (2D‐LC) demonstrated great resolving power for the separation of peptides, coupling with high precision tandem mass spectrometry (MS‐MS), making the throughput scale of deep proteome to thousands of proteins (Wang, Xiao, Liu, et al, ). However, this 2D‐LC‐MS/MS analysis strategy has been less frequently applied to the study of meat proteomes.…”