Diverse range of small peptides associated with high-density lipoprotein

Abstract

High-density lipoproteins (HDL) were examined as potential carriers of small peptides in plasma. HDL purified by density gradient centrifugation was delipidated and fractionated by size-exclusion chromatography under denaturing conditions. By hplc and mass spectrometry, more than 100 peptide components were found in the size range from 1,000 to 5,000 Da. By sequence analysis, peptides were identified as fragments of proteins such as apolipoproteins, fibrinogen, α 1 -proteinase inhibitor, and transthyretin. The results indicate that purified HDL bears a complex range of small peptides. It is unclear whether the peptides have any significant functional role as apolipopeptides, but they may represent a pathway for peptide delivery or scavenging and a significant reservoir of plasma peptides for diagnostic evaluation. KeywordsHigh-density lipoprotein; Apolipoprotein; Peptidomics; Transthyretin; Apolipoprotein A-I; Proteomics Abbreviations HDL, high-density lipoprotein; MALDI TOF MS, matrix-assisted laser desorption time-of-flight mass spectrometry; m/z, mass/charge ratio Lipoprotein particles in the blood circulation have multiple physiological roles as carrier molecules. In addition to the well recognized role of lipoproteins in cholesterol and triglyceride transport [1,2], lipoprotein particles including high-density, low-density, and very low-density lipoproteins (HDL, LDL, and VLDL) serve as carriers for other lipids and for fat-soluble vitamins such as vitamins A, D, E, K, and carotenoids [3,4]. A number of well-characterized proteins are major structural and functional components of HDL, LDL, and VLDL including apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, and E [1]. Other molecules without direct roles in lipoprotein metabolism such as endotoxin [5], tissue factor pathway inhibitor [6], paraoxonase [7], and ghrelin [8] also bind to HDL. The diversity of molecules binding to lipoprotein particles suggests that they can serve as a versatile adsorptive phase, analogous to stationary phases in reverse-phase chromatography. This characteristic led to a hypothesis that lipoproteins might serve as a reservoir for the hundreds of small peptides that circulate in human plasma [9,10]. The complex pattern of plasma peptides contains large amounts of potential information, and some studies suggest this information may be probed for cancer detection or other diagnostic purposes [11,12]. Initial promise of this approach has been slowed by reproducibility and standardization issues [13,14]. Most of the small peptides in plasma or serum now are considered to circulate bound to albumin and subfractionation of binding components may improve analyses [15][16][17]. Here, we examine whether HDL serves as a major carrier of small peptides. Materials and MethodsCalibrators and α-cyano-4-hydroxycinnamic acid and sinapinic acid were from Bruker Daltonics (Billerica, MA HDL was prepared from 400 mL of blood freshly collected in citrate preservative from a healthy donor. Plasma was separated from cells by centrifugation an...

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

“…However, the composition of the peptides in urine is not clearly understood. The lack of analysis of peptides in urine is likely due to the technical difficulty of measuring peptides in the presence of proteins [17]. Singh et al [43] has studied the variety of peptides in urine from patients with renal disease by combined Lowry method (which detects both proteins and peptides) and Bradford method (which detects only proteins) together.…”

“…The results showed that the excretion of peptides decreased in the presence of proteinuric renal disease [43]. However, this method was controversial because other contaminants such as urinary phenols, free amino acids, other endogenous compounds, and common drugs could interfere with the assay [17]. It is well known that urine contains high concentrations of small metabolites such as nucleotides and organic compounds.…”

“…Peptidome, which is defined as the low molecular weight (LMW) portion of proteome (MW < 15, 000 Da) [14][15][16], has attracted increasing attention in recent years [17][18][19]. Most reports are focused on the peptidome of serum/plasma because they are most commonly used in clinical diagnosis and contain high content of peptides/proteins [20][21][22].…”

“…Most reports are focused on the peptidome of serum/plasma because they are most commonly used in clinical diagnosis and contain high content of peptides/proteins [20][21][22]. Cancer diagnosis has been successfully achieved by the profiling of peptides in serum [17,[23][24][25]. Petricoin et al [26] first reported the diagnosis of ovarian cancer by using SELDI-TOF MS to profiling of the serum peptidome.…”

“…Solid phase extraction (SPE) is currently the most commonly used method to extract the peptides from urine [28][29][30][31], and is mainly based on their hydrophobicity but has no size selectivity. Nanoporous materials have shown great promise for sample pretreatment of body fluid in recent years [17,34,35]. The silica materials with highly ordered mesoporous structure such as M41s [36] and SBA-15 [37] have unique properties of high surface areas, extremely narrow pore size distribution and perfectly adjustable pore size.…”