scholarly journals Systematic examination of protein extraction, proteolytic glycopeptide enrichment and MS/MS fragmentation techniques for site-specific profiling of human milk N-glycoproteins

Talanta ◽  
2021 ◽  
Vol 224 ◽  
pp. 121811
Author(s):  
Bum Jin Kim ◽  
David C. Dallas
Keyword(s):  
Human Milk ◽  
Protein Extraction ◽  
Site Specific ◽  
Glycopeptide Enrichment ◽  
Systematic Examination ◽  
Fragmentation Techniques

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

2009 ◽  
Vol 395 (2) ◽  
pp. 178-188 ◽  
Author(s):  
Jessica Wohlgemuth ◽  
Michael Karas ◽  
Thomas Eichhorn ◽  
Robertus Hendriks ◽  
Sven Andrecht
Keyword(s):  
Protein Glycosylation ◽  
Site Specific ◽  
Glycopeptide Enrichment ◽  
Specific Analysis

scholarly journals Human Milk Proteins and Their Glycosylation Exhibit Quantitative Dynamic Variations during Lactation

2019 ◽  
Vol 149 (8) ◽  
pp. 1317-1325 ◽  
Author(s):  
Elisha Goonatilleke ◽  
Jincui Huang ◽  
Gege Xu ◽  
Lauren Wu ◽  
Jennifer T Smilowitz ◽  
...  
Keyword(s):  
Human Milk ◽  
Immunoglobulin A ◽  
Milk Proteins ◽  
Structure Stability ◽  
Mature Milk ◽  
Site Specific ◽  
Quantitative Changes ◽  
Protein Structure Stability ◽  
Analytical Tools ◽  
Functional Components

ABSTRACTBackgroundProteins in human milk are essential and known to support the growth, development, protection, and health of the newborn. These proteins are highly modified by glycans that are currently being recognized as vital to protein structure, stability, function, and health of the intestinal mucosa. Although milk proteins have been studied, the quantitative changes in milk proteins and their respective site-specific glycosylation are unknown.ObjectiveThis study expanded the analytical tools for milk proteins and their site-specific glycosylation and applied these tools to a large cohort to determine changes in individual protein concentrations and their site-specific N-glycosylation across lactation.DesignA tandem mass spectrometry method was applied to 231 breast-milk samples from 33 mothers in Davis, California, obtained during 7 different periods of lactation. Dynamic changes in the absolute abundances of milk proteins, as well as variation in site-specific N-glycosylation of individual proteins, were quantified.Resultsα-Lactalbumin, β-casein, k-casein, and α-antitrypsin were significantly increased from colostrum to transitional milk (4.37 ± 1.33 g/L to 6.41 ± 0.72 g/L, 2.25 ± 0.86 g/L to 2.59 ± 0.78 g/L, 1.33 ± 0.44 g/L to 1.60 ± 0.39 g/L, and 0.09 ± 0.10 g/L to 0.11 ± 0.04 g/L, respectively; P < 0.002). α-Lactalbumin (37%), β-casein (9%), and lysozyme (159%) were higher in mature milk than in colostrum. Glycans exhibited different behavior. Fucosylated glycans of lactoferrin and high-mannose, undecorated, fucosylated, sialylated, and combined fucosylated + sialylated glycans of secretory immunoglobulin A increased during lactation even when the concentrations of the parent proteins decreased.ConclusionsProteins in healthy mothers vary dynamically through lactation to support the development of infants. Individual milk proteins carried unique glycan modifications that varied systematically in structure even with site specificity. The role of glycosylation in human milk proteins will be important in understanding the functional components of human milk. This trial was registered at clinicaltrials.gov as NCT01817127.

scholarly journals IgGs from Human Milk Hydrolyze microRNAs

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2366
Author(s):  
Ivan Yu. Kompaneets ◽  
Evgeny A. Ermakov ◽  
Sergey E. Sedykh ◽  
Valentina N. Buneva ◽  
Georgy A. Nevinsky
Keyword(s):  
Human Milk ◽  
Biological Fluids ◽  
Catalytic Antibodies ◽  
Bioactive Components ◽  
Site Specific ◽  
Mother's Milk ◽  
Mother’S Milk ◽  
Hydrolysis Of ◽  
Breast Fed ◽  
Average Individual

Mother’s milk provides breast-fed infants with various nutrients, including peptides, proteins, DNA, RNA, antibodies, and other bioactive components promoting neonatal growth and protecting infants from viral and bacterial infection. The functions of many human milk components regarding the nutrition and protection of newborns may be very different compared to those of various biological fluids of healthy adults. For example, human milk contains catalytic antibodies (abzymes) with protein, lipid, and oligosaccharide kinase activities, which are absent in the biological fluids of healthy people and autoimmune patients. Obviously, the nutrition of infants with fresh breast milk is a special phenomenon having a very specific and important role. Here, we have shown that mother’s milk IgGs effectively split homo-(pN)23, and four miRNAs: miR-137, miR-219a-5p, miR-219-2-3p, and miR-9-5p. It was shown that ribonuclease activity is a unique property of milk IgGs. On average, individual IgGs hydrolyze (pA)23, (pU)23, and (pC)23 nonspecifically and with comparable efficiency, whereas the hydrolysis of four miRNAs is predominately site-specific. The specific sites of the hydrolysis of four miRNAs by IgGs from the blood of schizophrenic (SCZ) patients and secretory immunoglobulins A (sIgAs) from human milk were found earlier. The sites of the hydrolysis of four miRNAs by milk IgGs and sIgA-abzymes are almost the same, but are significantly different in comparison with those for SCZ IgGs. In addition, in contrast to the SCZ IgGs, milk IgGs and sIgAs efficiently hydrolyzed miRNAs in the duplex regions formed by their terminal sequences.

scholarly journals Quantitative Longitudinal Inventory of the N-Glycoproteome of Human Milk from a Single Donor Reveals the Highly Variable Repertoire and Dynamic Site-Specific Changes

2020 ◽  
Vol 19 (5) ◽  
pp. 1941-1952
Author(s):  
Jing Zhu ◽  
Yu-Hsien Lin ◽  
Kelly A. Dingess ◽  
Marko Mank ◽  
Bernd Stahl ◽  
...  
Keyword(s):  
Human Milk ◽  
Site Specific ◽  
Single Donor

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

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