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4D Label-free quantitative proteomic identification and analysis of protein components in plasma derived and recombinant Human Serum Albumin preparations

ABSTRACT: Clinical therapeutic human serum albumin (HSA) preparations are manufactured by human plasma from donors and contain some miscellaneous proteins (MPs) except albumin. Multiple studies have demonstrated significant variations in the extent of post-translation modifications, redox states, and antioxidant capacities among HSA preparations from different manufacturers. However, these studies have primarily focused on the albumin molecule itself and have not conducted detailed analysis and comparison of the MPs present in HSA preparations. While there have been a few studies that have reported on the presence of MPs in commercial HSA preparations, research in this area is still relatively scarce. In this study, we employed 4D Label-Free quantitative proteomic technology to conduct identification and analysis of MPs in preparations of human plasma-derived HSA (pHSA) from 6 distinct manufacturers; Additionally, we investigated MPs in recombinant HSA (rHSA) preparations obtained through expression in yeast and rice. There were total 456 different MPs identified in 6 pHSA samples and 96 MPs were detected in all pHSA samples. 52 MPs in the rHSA (expressed in yeast) sample and 152 in the rHSA (expressed in rice) were identified. Haptoglobin, Hemopexin and Transthyretin included the top 8 MPs with the highest relative abundance detected in every pHSA preparations in this study. Bioinformatics (including GO, COG/KOG, KEGG, protein−protein interactions, etc.) analysis showed that the MPs identified in pHSA were basically involved in endopeptidase inhibitor activity, complement and coagulation cascades, biosynthesis of amino acids and cholesterol metabolism. Validation results demonstrated that HP, HPX, TF and TTR could be detected in pHSA but not in rHSA, which were consistent with 4D Label-Free quantitative proteomic results. Compared with previous studies, more trace MPs were detected in pHSA and rHSA preparations by 4D Label-Free quantitative proteomic technology. This could provide a new approach for high-throughput detection of trace MPs in HSA preparations.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Oryza Sativa (rice) Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Blood

SUBMITTER: jun hou

LAB HEAD: Qing Liu

PROVIDER: PXD045339 | Pride | 2025-06-03

REPOSITORIES: Pride

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Project description:This study aims to analyze and compare the post-translational modification differences between human plasma- and genetic recombination-derived human serum albumin (HSA) in clinical treatments in China. 6 different post-translational modifications, including acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation, were detected using pan-specific antibodies through Western blot. The samples, consisting of human plasma-derived HSA (pHSA) from six different manufacturers and recombinant HSA (rHSA) expressed in yeast and oryza sativa, underwent detection of types of post-translational modifications. 4D label-free PTMs quantitative proteomic analysis was conducted to detect N-glycosylation and the above-mentioned post-translational modifications in pHSA and rHSA samples, and analyze the differences in modification sites and levels. Through Western blot analysis, all six pHSA and two rHSA samples were positive in the band of albumin (66.5 kDa) for the six post-translational modifications. Further analysis using 4D label-free PTMs quantitative proteomics revealed 25 (29) acetylated, 30 (32) succinylated, 41 (50) malonylated, 15 (23) phosphorylated, 36 (30) beta-hydroxybutyrylated, and 27 (34) lactylated modification sites in pHSA and rHSA samples and no N-glycosylation modification sites. The analysis results identified 1 acetylation (ALB_K160), 2 beta-hydroxybutyrylation (ALB_K569, ALB_K426), 3 crotonylation (ALB_K264, ALB_K581, ALB_K560), and 15 phosphorylation specific modification sites in pHSA, as well as 3 crotonylation (ALB_K560, ALB_K562, ALB_K75), 1 succinylation (ALB_K490), and 23 phosphorylation specific modification sites in rHSA. In pHSA (rHSA), 2 (6) acetylation, 10 (12) succinylation, 0 (9) crotonylation, 1 (9) phosphorylation, 6 (0) beta-hydroxybutyrylation, and 0 (7) lactylation specific modification sites were found. Additionally, in the common modification sites of pHSA and rHSA, pHSA showed up-regulation of amberylation (16:1) and beta-hydroxybutyrylation (12:2) in more sites, and up-regulation of acetylation (7:11), crotonylation (2:11), phosphorylation (1:8), and lactylation (1:14) in less sites compared to rHSA. In clinical treatment, the used pHSA and rHSA in China generally exhibit acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation, and there are differences in the site characteristics and modification levels of these modifications between pHSA and rHSA. Further experimental investigations are needed to explore the impact of post-translational modification differences on the biological function, efficacy, and safety of pHSA and rHSA.

Project description:Background Plasma proteins can be modified by addition of sugar residues by non-enzymatic glycation as well as glycosylation. While glycation is a hallmark of hyperglycemia, glycosylation is a complex, enzyme-catalyzed post-translational modification by heterogeneous sugar chains and is present on a majority of plasma proteins. N-linked glycosylation occurs on asparagine residues occurring predominantly on a canonical N-glycosylation motif (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported to be glycosylated less frequently. Albumin is the most abundant protein in human plasma whose glycation has been implicated in end-organ damage in advanced diabetes mellitus, and as such, has both diagnostic and therapeutic implications. However, albumin has long been regarded as a non-glycosylated protein owing to the absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated with two possible attached glycans. Methods To investigate N-linked glycosylation of abundant serum proteins in greater detail, we passed healthy donor samples through a multiple affinity removal spin (MARS14) column followed by trypsin digestion of bound proteins and subsequent glycopeptide enrichment by size-exclusion chromatography (SEC) or mixed-mode anion-exchange (MAX), in parallel. Global analysis of intact glycopeptides was performed by LC-MS/MS to evaluate potential glycosylation at canonical as well as non-canonical sites. PNGase F treatment was carried out to confirm N-linked glycosylation at non-canonical sites Asn-X-Cys/Val. Glycopeptides on albumin at non-canonical sites were further characterized by MS3 fragmentation from immunoprecipitated albumin to confirm the attached glycans. To assess if the observed glycosylation was a general phenomenon, targeted analysis using parallel reaction monitoring (PRM) was carried out on an independent set of twenty human serum samples. Finally, to test whether albumin glycosylation is unique to human albumin or is a general phenomenon, bovine and rabbit serum albumins were subjected to MAX chromatography and LC-MS/MS analysis. Results We report that human albumin is modified by N-linked glycosylation at two sites, Asn68(-Glu-Val) and Asn123-(Glu-Cys), both of which occur in non-canonical N-glycosylation motifs. We detected N-glycopeptides at both sites bearing four complex mono- and bi-antennary N-linked glycans, with Hex5HexNAc4NeuAc2 and Hex5HexNAc4NeuAc1 being the most abundant glycans. These findings were validated by analyzing deglycosylated peptides and MS3-based fragmentation of glycopeptides. Glycosylation at both sites was confirmed by targeted MS in twenty donor samples. Finally, we report that the Asn residue on bovine and rabbit serum albumins that corresponds to the highly conserved Asn123 in human albumin is also N-glycosylated with complex sialylated glycans similar to those observed in human albumin. Conclusions Albumin is a conserved glycoprotein with multiple N-linked glycosylation sites. This glycosylation of albumin has potential functional implications and applications in medicine.

Project description:Study question: Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? Summary answer: A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable. What is known already: There are no data in the literature on what non-declared proteins are present in un-conditioned (fresh media in which no embryos have been cultured) commercial embryo media. Study design, size, duration: The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analysed for each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analysed. Participants/materials, setting, methods: For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulphate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Main results and the role of chance: Using advanced mass spectrometry and high confidence criteria for accepting proteins (p< 0.01), a total of 110 proteins other than HSA were identified. The average serum albumin content was found to be 94% (92% - 97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. Limitations, reasons for caution: For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. Wider implications of the findings: The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analysing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in un-conditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring.

Project description:Investigation of whole genome gene expression level changes in BMDM adhered for 4 or 18hrs to Lab Tek Chamber slides pre-coated with 4ug/ml of human serum albumin (HSA) (control protein) or purified human C1q and treated with lipopolysaccharide (LPS: 20ng/ml) Mycobacterium avium 101 (M avium) or apoptotic Jurkat T cells. BMDM were obtained from C57Bl/6 and generated as previously described in Bohlson, SS, Strasser, JA, Bower JJ, Schorey J S 2001 Role of complement in Mycobacterium avium pathogenesis: in vivo and in vitro analyses of the host response to infection in the absence of complement component C3 Infec Immun 69: 7729-7735. Mybacterium avium 101 was obtained from Dr Jeff Schorey (University of Notre Dame). The human Jurkat T cell line was obtained from ATCC (Manassas, VA) and induced to undergo apoptosis with dexamethasone as described in Lillis, AP, Greenlee, M C, Mikhailenko I, Pizzo, S V, Tenner, A J, Strickland, D K, Bohlson, S S 2008 Murine Low-Density Lipoprotein Receptor-Related Protein 1 (LRP) Is Required for Phagocytosis of Targets Bearing LRP Ligands but Is Not Required for C1q-triggered Enhancement of Phagocytosis J Immunol 181: 364: 373. A six chip study using total RNA from 5 separate cultures of BMDM adhered to HSA for 4 hrs, 5 separate cultures of BMDM adhered to C1q for 4 hrs, 5 separate cultures of BMDM adhered to HSA for 4 hrs and infected with M avium at a 1:500 ratio of BMDM to M avium, 5 separate cultures of BMDM adhered to C1q for 4hrs and infected with M avium at a 1:500 ratio ofBMDM to M avium, 5 separate cultures of BMDM adhered to HSA for 4 hrs and infected with M avium at a 1:1000 ratio of BMDM to M avium, 5 separate cultures of BMDM adhered to HSA for 4 hrs and treated with 20ng/ml LPS, 5 separate cultures of BMDM adhered to C1q for 4 hrs and treated with 20ng/ml LPS, 5 separate cultures of BMDM adhered to HSA for 4 hrs and treated with 20ng/ml LPS and co-cultured with apoptotic Jurkat cells at a 1:3 ratio of BMDM to apoptotic cells, 5 separate cultures of BMDM adhered to C1q for 4 hrs and treated with 20ng/ml LPS and co-cultured with apoptotic Jurkat cells at a 1:3 ratio of BMDM to apoptotic cells, 5 separate cultures of BMDM adhered to HSA for 18 hrs, 5 separate cultures of BMDM adhered to C1q for 18 hrs, 5 separate cultures of BMDM adhered to HSA for 18 hrs and infected with M avium at a 1:500 ratio of BMDM to M avium, 5 separate cultures of BMDM adhered to C1q for 18hrs and infected with M avium at a 1:500 ratio of BMDM to M avium, 5 separate cultures of BMDM adhered to HSA for 18 hrs and infected with M avium at a 1:1000 ratio of BMDM to M avium. The chip utilized for these studies is a mouse whole-genome 12-plex expression microarray design by NimbleGen designed from the MM9 genome Candidate probe sequences were verified to have no cross-hybridization to human (HG19) or Mycobacterium avium (NC_008595) targets Note: Study sample BMDM_HSA & M avium (1:500)_18hr_rep2 was not included in this submission due to quality control concerns.

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4D Label-free quantitative proteomic identification and analysis of protein components in plasma derived and recombinant Human Serum Albumin preparations

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