title: Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion.
abstract: A formic acid (FA)-assisted sample preparation method was presented for protein identification via mass spectrometry (MS). Detailedly, an aqueous solution containing 2% FA and dithiothreitol was selected to perform protein denaturation, aspartic acid (D) sites cleavage and disulfide linkages reduction simultaneously at 108°C for 2h. Subsequently, FA wiped off via vacuum concentration. Finally, iodoacetamide (IAA) alkylation and trypsin digestion could be performed ordinally. A series of model proteins (BSA, β-lactoglobulin and apo-Transferrin) were treated respectively using such method, followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The identified peptide number was increased by ∼ 80% in comparison with the conventional urea-assisted sample preparation method. Moreover, BSA identification was achieved efficiently down to femtomole (25 ± 0 sequence coverage and 16 ± 1 peptides) via such method. In contrast, there were not peptides identified confidently via the urea-assisted method before desalination via the C18 zip tip. The absence of urea in this sample preparation method was an advantage for the more favorable digestion and MALDI-TOF MS analysis. The performances of two methods for the real sample (rat liver proteome) were also compared, followed by a nanoflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry system analysis. As a result, 1335 ± 43 peptides were identified confidently (false discovery rate <1%) via FA-assisted method, corresponding to 295 ± 12 proteins (of top match=1 and requiring 2 unique peptides at least). In contrast, there were only 1107 ± 16 peptides (corresponding to 231 ± 10 proteins) obtained from the conventional urea-assisted method. It was serving as a more efficient protein sample preparation method for researching specific proteomes better, and providing assistance to develop other proteomics analysis methods, such as, peptide quantitative analysis.
authors: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors: Shuaibin
authors: S
authors: Wu
authors: Kaiguang
authors: K
authors: Yang
authors: Zhen
authors: Z
authors: Liang
authors: Lihua
authors: L
authors: Zhang
authors: Yukui
authors: Y
authors: Zhang
mesh_terms: D000818:Animals; D002417:Cattle; D005561:Formates / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods* / Q000592:standards; D014357:Trypsin / Q000378:metabolism*; D014508:Urea / Q000737:chemistry
chemical_list: D005561:Formates; D011506:Proteins; C030544:formic acid; D014508:Urea; D014357:Trypsin