Global Survey of Mouse Liver Protein Expression Using Liquid Isoelectric Focusing Prefractionation of Tryptic Peptides and LC-MS/MS†

doi:10.7503/cjcu20141120

Abstract

Abstract:

To comprehensively and quickly identify proteins of mouse liver, the tryptic peptides were pre-fractioned and enriched by isoelectric focusing technigue using Agilent 3100 OFFGEL Fractionator into 24 fractions, followed by RPLC and MS/MS analysis. Each MS/MS spectrum was searched for mouse species against the Swissprot database. The results were analyzed which resulted in the identification of proteins in mouse liver. Finally, 3474 proteins were identified from mouse liver, 832 of them were new identified proteins. Bioinformatic analyses were performed to demonstrate the physicochemical properties, cellular locations and functions of the proteins. The combined use of liquid fractionation and LC-MS allows a considerable increase in proteome coverage of very complex samples prepared from the tissues extracts and supports the low-concentrated protein identification. This study can provide useful data to research C57 mouse liver proteins.

Key words: Proteomics, Liquid isoelectric focusing, LC-MS/MS, Mouse liver protein

CLC Number:

O657

TrendMD:

LI Ying, YAO Jun, YANG Pengyuan, FAN Huizhi. Global Survey of Mouse Liver Protein Expression Using Liquid Isoelectric Focusing Prefractionation of Tryptic Peptides and LC-MS/MS^†[J]. Chem. J. Chinese Universities, 2015, 36(6): 1074.

Figures/Tables 9

Fig.1 Analysis workflow used to profile mouse liver proteins

Fig.2 Number distribution of the identified peptides in OFFGEL fraction wells(A) and fraction distribution of the identified proteins(B)

Fig.3 Cellular components GO mapping of the identified proteins(A) and biological process GO mapping of the identified proteins(B)

Table 1 Number of protein identification classified by number of peptides used for identifications

Protein number	Protein number of four or more unique peptide	Protein number of three unique peptide	Protein number of two unique peptide
3474	2433(70%)	378(11%)	663(19%)
7099^[20]	2857(40%)^[20]	1475(21%)^[20]	2758(39%)^[20]

Fig.4 Comparison of four mouse liver proteomes

Table 2 Distribution of abundance of proteins(iBAQ intensities) identified with FDR of 1%

Protein number	Number of iBAQ intensities 1.0×10⁹—1.0×10⁷ of protein	Number of iBAQ intensities 1.0×10⁷—1.0×10⁵ of protein	Number of iBAQ intensities 1.0×10⁵—1.0×10³ of protein
3474^a	470(13.5%)	2312(66.5%)	692(20%)
832^b	8(1%)	527(63%)	299(36%)

Fig.5 Cellular components GO mapping of new identified proteins(A) and KEGG pathways mapping of new identified proteins(B)

Fig.6 Distribution of peptides of new proteins identified in relation to their theoretical molecular mass(A), pI(B) and GRAVY values(C)(A) Molecular weight: a. 700—1000; b. 1100—1500; c. 1500—1900; d. 1900—2300; e. 2300—2700; f. 2700—3100; g. >3100. (B) pI: a. 3—4; b. 4—5; c. 5—6; d. 6—7; e. 7—8; f. 8—9; g. 9—10; h. 10—11; i. 11—12; j. 12—13; (C) GRAVY value: a. >0; b. -0.25—0; c. -0.5—0.25; d. -0.75—-0.5; e. -1.00—-0.75; f. <-1.00.

Fig.7 TIC of peptide fraction eluted(A) and MS spectrum(inset) of peptides effluent(at 108.2 min) labled by ★ in (A) and MS/MS spectrum(B) for precursor ion of m/z 685.20 labled by ★ in the inset of (A)

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