Comprehensive Peptidome Analysis of Mouse Livers by Size Exclusion Chromatography Prefractionation and NanoLC−MS/MS Identification

Abstract

Peptidome analysis has received increasing attention in recent years. Cancer diagnosis by serum peptidome has also been reported by peptides' profiling for discovery of peptide biomarkers. Tissue, which may have a higher biomarker concentration than blood, has not been investigated extensively by means of peptidome analysis. Here, a method for the peptidome analysis of mouse liver was developed by the combination of size exclusion chromatography (SEC) prefractionation with nanoliquid chromatography-tamdem mass spectrometry (nanoLC-MS/MS) analysis. The extracted peptides from mouse liver were separated according to their molecular weight using a size exclusion column. MALDI-TOF MS was used to characterize the molecular weight distribution of the peptides in fractions eluted from the SEC column. The low molecular weight (LMW) (MW < 3000 Da) peptides in the collected fractions were directly analyzed by LC-MS/MS which resulted in the identification of 1181 unique peptides (from 371 proteins). The high molecular weight (HMW) (MW > 3000 Da) peptides in the early two fractions from the SEC column were first digested with trypsin, and the resulted digests were then analyzed by LC-MS/MS, which led to the identification of 123 and 127 progenitor proteins of the HMW peptides in fractions 1 and 2, respectively. Analysis of the peptides' cleavage sites showed that the peptides are cleaved in regulation, which may reflect the protease activity and distribution in body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery.

Analysis of the human urine endogenous peptides by nanoparticle extraction and mass spectrometry identification

“…The identified number of the peptides was not as high as other reports, such as from the serum/plasma [21,39] or tissue peptidome [48]. The reasons for phenomenon probably are: (a) the peptide amounts in urine are actually low because the kidney will reabsorb some peptides/proteins released into the urine.…”

“…Size exclusion chromatography (SEC) is known to be able to separate samples according to their molecular weight based on the size sieving mechanism, which makes it easier to identify the peptides by MS/MS. We have reported previously that low molecular weight peptides can be efficiently separated by SEC by using mobile phase without salt [48]. The SEC have very good reproducibility compared to other chromatographic separations due to its pure separation according the molecular weight of the analytes [48].…”

“…We have reported previously that low molecular weight peptides can be efficiently separated by SEC by using mobile phase without salt [48]. The SEC have very good reproducibility compared to other chromatographic separations due to its pure separation according the molecular weight of the analytes [48]. Here, we used a size exclusion column to separate the extracted peptides into fractions.…”

“…Majority of our identified peptides had a molecular weight below 3500 Da, though there were also a significant number of peptides with HMW. So far, we can only get the molecular weight of the HMW peptides by MALDI-TOF MS or identify their progenitor protein by trypsin digestion [48]. With further development of MS technology such as TOF-MS [49] and FT-MS [50], the identification of the HMW peptides will be become more accessible.…”

Automation of nanoflow liquid chromatography‐tandem mass spectrometry for proteome and peptide profiling analysis by using a monolithic analytical capillary column

“…The procedure of peptide sample extracted from mouse liver was identical with our previous report [23]. Briefly, mouse livers were homogenized and sonicated.…”