Title : Glycansfrom
HDL Com
position ally Profiled by Nano-LC–MS. TheN-glycans were released using the protocol described in Experimental Procedures
Abstract :
- Analyses were done fromthe isolated HDL triplicates
- The glycan profile of HDL is illustratedwith one of the triplicates (Figure 2)
- Thedepicted glycan structures were based on biological precedence andtheir correct masses; however, isomers are putative
- HPLC-microchip packed with a PGC stationary phase was usedto separate glycans with high sensitivity and reproducibility
- The total ion chromatogram (TIC) from the raw LC–MS datawas separated into a number of extracted compound chromatograms (ECCs)
- The extractor algorithm was based on the expected charge carriers,potential neutral mass losses, and a predicted isotopic distribution
- Each peak represents one compound eluting at a certain retention timewith the detected m/z, which enablesthe assignments of glycan com position
- The most abundant glycan (includingisomers) was a biantennarycomplex type glycan with two sialic acids (Hexose5HexNAc4Neu5Ac2)
- Perhaps not surprisingly, these glycansare also the most abundant in blood
- Inthis regard, HDL-associated proteins are similar to other abundantglycoproteins in blood
- Two abundantisomers were observed eluting at retention times 23 and 25 min
- Mostof the glycans (90%) from HDL glycoproteins were sialylated with oneor two Neu5Ac(s)
- Ion intensities were measured in the MS mode basedon the protonated molecular ion in positive mode (Table 1)
- Following the most abundant glycan with two Neu5Ac , a complextype biantennary glycan with one Neu5Ac was shown in a high intensityeluting at 17–20 min including four observed isomers
- Our groupreported glycans with sialic acids are retained longer with PGC
- Here we also observed that the sole additionof an acidic NeuAcresidue increased the retention time by nearly 5 min
- Sialic acid , mostly found as a terminal componentof glycoproteinsand glycolipids on the outer surface of cells, is involved in cellularsecretions
- Sialic acid participatesin multiple and diverse cellular events, such as acting as an antirecognitionagent by shielding recognition sites and conversely by beinga biological recognition site as a ligand for multiple molecules
- Sialic acid is anionic and therefore likely contributes to HDL ’snegative charge
- Previously, it has been shown that the addition ofphosphatidylinositol, which increases HDL ’s negative charge,blocked binding of HDL to hepatic lipase
- Other previous studies have also shown that the electronegativityof HDL affects its binding to cholesterol ester transfer protein andthus the exchange of cholesterol esters with LDL
- The sialylated glycans in HDL associated proteins may beinvolved in molecular and cellular interactions that affect the functionof HDL
- For example, sialic acid residues of ApoE may be essentialfor its recognition by HDL3 particles and further the bindingto HDL3
- Interestingly, desialylationof lipoprotein-associated apoproteins was found to be associated withincreased circulating neuraminidase excreted by Streptococcuspneumonia in patients with hemolytic uremic syndrome, suggesting that bacterial infection may leadto desialylation of HDL particles in vivo
- In addition to complex-typesialylated N-glycans, fucosylated N-glycanswere present at relatively low abundances
- High-mannose and hybridtype N-glycans were also observed, indicating the variety of N-glycansfrom HDL-associated proteins
- The wide dynamic range of the detectedN-glycans by the described method allows the evaluation of all typesof oligosaccharides and further analyses of their biological functions
- Supplemental Figure S1, Supporting Information shows the overlaid ECCs of N-glycans from the three replicates
- The separation of glycans and their profiles was similar, as the mostabundant ones were observed as sialylated complex-type eluting at23–25 min
- The method is considered reproducible with regardto the glycan com position assignments from these technical triplicates
Output (sent_index, trigger,
protein,
sugar,
site):
- 10. abundantglycoproteins, , abundantglycoproteins, -, -
- 12. glycoproteins, , HDL glycoproteins, -, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):