About

Stacy A. Malaker, Ph.D., is the Principal Investigator of the Malaker Lab, where her research focuses on glycoproteomics, including the development and optimization of protocols, instrumentation, and programs for studying glycoproteins. Her background includes working on mass spectrometric analysis of energetic materials during her B.S. studies and improving methodologies for mass spectrometric identification of post-translationally modified antigens during her Ph.D. at the University of Virginia. Her work aims to advance understanding of glycosylation and its role in biological systems, particularly in relation to disease states such as cancer. Dr. Malaker's research integrates mass spectrometry techniques to explore the structure and function of glycoproteins, with a focus on mucin-domain glycoproteins and their implications in health and disease.

Research topics

• Biology
• Cell biology
• Biochemistry
• Computational biology
• Immunology
• Genetics
• Chemistry
• Internal medicine
• Organic chemistry
• Cancer research
• Chromatography
• Microbiology
• Medicine

Selected publications

• Characterization of O-glycoproteases for applications in mass spectrometry

  Methods in enzymology on CD-ROM/Methods in enzymology · 2026-01-01

  book-chapterSenior author
  PublisherDOI

• Erratum: Decoding Extracellular Protein Glycosylation in Human Health and Disease

  Annual Review of Analytical Chemistry · 2026-01-07

  articleSenior author
  PublisherDOI

• Impaired glycosylation promotes rapid transition to hepatocellular carcinoma in model of diet-induced steatotic liver disease

  Journal of Clinical Investigation · 2026-03-10

  articleOpen access

  Obesity-linked steatosis is a significant risk factor for hepatocellular carcinoma (HCC); however, the molecular mechanisms underlying the transition from metabolic dysfunction-associated steatotic liver disease (MASLD) to HCC remain unclear. Here, we explored the role of the ER-associated protein NgBR, an essential component of the cis-prenyltransferase (cis-PTase) enzyme, in chronic liver disease. Hepatocyte-specific NgBR deletion in mice (N-LKO) intensified triacylglycerol (TAG) accumulation, inflammatory responses, ER/oxidative stress, and fibrosis, ultimately resulting in HCC development with 100% penetrance after 4 months on a high-fat diet. Similarly, liver-specific knockout of DHDDS, NgBR's cis-PTase partner, and a knockin model carrying a human NgBR mutation that impairs cis-PTase activity developed HCC under high-fat diet conditions, although with lower penetrance. A single-cell transcriptomic atlas from affected livers provides a detailed molecular analysis of the transition from liver pathophysiology to HCC development. Mechanistically, NgBR deficiency promoted excessive hepatic TAG accumulation by enhancing lipid uptake and impairing VLDL secretion. Importantly, pharmacological inhibition of diacylglycerol acyltransferase-2 (DGAT2), a key enzyme in TAG synthesis, abrogated diet-induced liver damage and HCC burden in N-LKO mice. Overall, our findings establish cis-PTase as a critical suppressor of MASLD-HCC conversion and suggest DGAT2 inhibition may serve as a promising therapeutic approach to delay HCC formation in advanced metabolic dysfunction-associated steatohepatitis.

  PublisherOA PDFDOI

• O-glycosylation affects proteolysis of the native tear peptidome

  ChemRxiv · 2026-01-07

  articleSenior author

  immune response and participating in antimicrobial activity. The biological function of these native peptides is governed by their biochemical properties including peptide length, charge, and hydrophobicity. Accordingly, the production of native peptides within the tear film is tightly regulated through a complex interplay between circulating proteases and the proteolytic susceptibility of the protein substrate. Though glycosylation has been shown to regulate the proteolysis of specific proteins in vitro, the extent to which endogenous cleavage motifs are mediated by proximal O-glycans remains unexplored in a more complex sample. Furthermore, the existence and identity of native tear peptides bearing O-glycans has yet to be elucidated, largely due to the high analytical complexity of tear fluid and the difficulty in characterizing O-glycosylated peptides. To address this gap, we leveraged advances in mass spectrometry (MS) to provide the first observation of circulating tear O-glycopeptides, detailing their biochemical properties and overall glycan compositions. Beyond systematic profiling of the O-glycopeptidome, we employed site-specific glycoproteomic analysis followed by molecular dynamics to investigate the interplay between O-glycan proximity and proteolysis. Here, we observed that O-glycans preferentially occupy glycosites distal from the N-/C-terminus, thereby influencing distinct cleavage motifs and peptide backbones. Finally, we showed in silico that O-glycosylation can mediate the solvent accessibility of proximal cleavage residues, thus providing a structural basis for these observations. Taken together, this study defines the proteolytic landscape at the ocular surface and highlights a potential role for tear fluid O-glycans.

  PublisherDOI

• Enrichment strategies for mucin-domain O-glycoproteins

  Methods in enzymology on CD-ROM/Methods in enzymology · 2026-01-01

  book-chapterSenior authorCorresponding
  PublisherDOI

• Xylosyltransferase engineering to manipulate proteoglycans in mammalian cells

  Nature Chemical Biology · 2026-01-20 · 1 citations

  articleOpen access

  Mammalian cells receive signaling instructions through interactions on their surfaces. Proteoglycans are critical to these interactions, carrying long glycosaminoglycans that recruit signaling molecules. Biosynthetic redundancy in the first glycosylation step by two xylosyltransferases XT1/2 complicates annotation of proteoglycans. Here we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. Through a bump-and-hole tactic, we engineer the two isoenzymes XT1 and XT2 to specifically transfer the chemically tagged xylose analog 6AzGlc to target proteins. The tag contains a bioorthogonal functionality, allowing to visualize and profile target proteins in mammalian cells. Unlike xylose analogs, 6AzGlc is amenable to cellular nucleotide-sugar biosynthesis, establishing the XT1/2 bump-and-hole tactic in cells. The approach allows pinpointing glycosylation sites by mass spectrometry and exploiting the chemical handle to manufacture proteoglycans with defined glycosaminoglycan chains for cellular applications. Engineered XT enzymes permit an orthogonal view into proteoglycan biology through conventional techniques in biochemistry.

  PublisherDOI

• Site-specific O-glycans influence lacritin structure and multimerization in tears

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-04-02

  articleOpen accessSenior authorCorresponding

  Lacritin is an abundantly expressed glycoprotein in tear fluid and plays key roles in immune response, tear secretion, and bacterial killing. These biological functions are tightly regulated through several biochemical mechanisms including multimerization, proteolysis, and alternative splicing, especially within its C-terminal domain. Given its critical role at the ocular surface, lacritin is currently under investigation as a diagnostic biomarker and therapeutic candidate for dry eye disease (DED). However, despite over three decades since its initial discovery, the functional significance of the O-glycans that comprise more than 50% of its molecular weight remain largely unknown. To address this gap, we leveraged mass spectrometry (MS)-based glycoproteomics and molecular dynamics (MD) to explore the structural role of site-specific O-glycans on C-terminal lacritin. In doing do, we identified distinct glycosylation profiles between monomeric and multimeric lacritin, particularly at glycosites located near crosslinking residues (Lys101 and Lys104) that modulate multimer formation. Building on our glycoproteomics data, we performed MD simulations on monomer and multimer glycoforms and revealed that O-glycans participate in intra-glycan-protein interactions, thereby affecting the conformational flexibility of lacritin and the spatial arrangement of Lys101 and Lys104. Finally, we quantified the solvent-accessible surface area (SASA) of Lys101 and Lys104, highlighting that proximal O-glycosylation is predicted to affect the propensity of these residues to participate in crosslinking. Taken together, these findings underscore a central role for lacritin O-glycans in affecting structural topology with implications for its downstream biological activity.

  PublisherOA PDFDOI

• Glycoproteoforms of Osteoarthritis-associated Lubricin in Plasma and Synovial Fluid

  Molecular & Cellular Proteomics · 2025-02-06 · 8 citations

  articleOpen access

  Lubricin/proteoglycan-4 (PRG-4) is a mucinous glycoprotein that lubricates cartilage and maintains normal tissue function and cell homeostasis. Altered O-glycoproteforms of lubricin have been found in osteoarthritis (OA) synovial fluid (SF), which could ostensibly be used to diagnose early onset OA. However, SF is invasive to obtain and generally would not be surveyed from otherwise healthy individuals. Thus, a plasma-based OA screening tool focused on lubricin glycosylation could be a less invasive method to aid in early-stage OA diagnosis. In this report, we used glycomics and glycoproteomics to characterize glycoproteoforms of OA lubricin in SF and plasma. We obtained near-complete sequence coverage of lubricin's mucin domain and its glycosylation using matched SF and plasma from patients with OA (N = 5). From SF lubricin we observed a spectrum of O-glycans ranging from a single GalNAcα1-Ser/Thr monosaccharide up to branched pentasaccharides. In contrast, plasma based lubricin was predominantly decorated with sialylated Galβ1-3GalNAcα1-Ser/Thr (Sialyl T). To explain the glycosylation differences observed between SF and plasma lubricin, we present splice variant-specific peptides found within the non-glycosylated region, revealing that that the longest spliceoform of lubricin was present exclusively in SF, while additional shorter splice variants could only be detected in plasma. Based on our glycoproteomic data, we developed and validated a lectin assay for lubricin, and applied this on a larger cohort of matched SF/plasma (N = 19) to confirm the glycosylation differences between SF and plasma proteoforms. Next, we leveraged our assay to screen over 100 patient with OA samples (OA patients N = 108/controls N = 38) to probe plasma lubricin as an OA biomarker. Here, we detected a decrease in α2,6 linked sialic acid in patients with OA and further show that the extent of α2,6 and α2,3 sialylation on plasma-associated lubricin correlated with patient characteristics, especially Body Mass Index (BMI).

  PublisherOA PDFDOI

• In-depth analysis of the tear fluid glycoproteome reveals diverse lacritin glycosylation and spliceoforms

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-18

  preprintOpen accessSenior authorCorresponding

  Tear fluid comprises a diverse group of extracellular glycoproteins which are critical for ocular homeostasis. Within the tear fluid glycoproteome, lacritin is highly expressed and plays a key role in immune response, tear secretion, and antimicrobial activity. Importantly, glycosylation constitutes over 50% of lactritin's molecular weight. However, despite this fact, nothing is known about the specific glycan structures on lacritin and how they influence its protein folding, function, or downstream biological processes. Similarly, it remains completely unknown whether alterations to lacritin glycans are correlated with ocular pathologies. To address this gap in knowledge, we harnessed mass spectrometry (MS) to conduct the first O-glycoproteomic study of tear fluid. Here, we report unprecedented coverage of lacritin glycosylation, detailing 19 O-glycosites bearing a myriad of glycan structures. Further, we leveraged Alphafold 3.0 and GlycoShape to visualize the impact of these glycans on its structure, demonstrating that O-glycosylation renders the protein backbone rigid and extended. Surprisingly, we also detected protein-level evidence of two lacritin spliceoforms, representing the first observation of these isoforms by MS. Simultaneously, we describe the most comprehensive characterization of the tear fluid glycoproteome to date, elucidating the glycosylation profile of Immunoglobulin A (IgA), lactoferrin, and other glycoproteins with demonstrated clinical relevance as diagnostic biomarkers. Overall, this study lays critical groundwork for future biochemical investigation of tear fluid glycoproteins and their application as diagnostic or therapeutic tools for ocular diseases.

  PublisherOA PDFDOI

• An ultrasensitive and modular platform to detect Siglec ligands and control immune cell function

  Science Advances · 2025-11-12 · 2 citations

  articleOpen access

  Siglecs are immunomodulatory receptors that regulate immune cell function. A fundamental challenge in studying Siglec-ligand interactions is the low affinity of Siglecs for their ligands. Inspired by how nature uses multivalency, we developed Siglec-liposomes as a highly multivalent and versatile platform for detecting Siglec glycan ligands in which recombinant Siglecs were conjugated to liposomes using the SpyCatcher-SpyTag system. Siglec-liposomes offer tunable multivalency and a modular assembly, enabling presentation of different Siglecs on the same liposome. Using Siglec-liposomes, we profiled Siglec ligands on human leukocytes, revealing distinct patterns of Siglec ligands. Moreover, Siglec-liposomes are in vivo compatible, where we demonstrated that Siglec-7-liposomes bind to the brain vasculature in a mucin domain-dependent manner. Given the abundance of Siglec ligands on T cells, we investigated whether Siglec-liposomes modulate T cell function and find that Siglec-7-liposomes increase T cell proliferation in an ST3Gal1-dependent and CD43-independent manner. Together, Siglec-liposomes are a versatile and sensitive tool for detecting Siglec ligands and immunomodulation.

  PublisherDOI

Recent grants

• Toward understanding the role of altered glycosylation in cancer

  NIH · $1.6M · 2022–2027

• An enzymatic approach to study cancer-associated cell-surface glycoproteins: exploration of mucin-degrading bacterial metalloproteases

  NIH · $125k · 2017–2019

Frequent coauthors

• Carolyn R. Bertozzi

  Stanford University

  98 shared

• Benjamin Schumann

  Imperial College London

  30 shared

• Isabelle Fournier

  Protéomique, Réponse Inflammatoire et Spectrométrie de Masse

  29 shared

• Michel Salzet

  Inserm

  29 shared

• Jusal Quanico

  University of Antwerp

  29 shared

• Kayvon Pedram

  Janelia Research Campus

  27 shared

• Antonella Raffo‐Romero

  Protéomique, Réponse Inflammatoire et Spectrométrie de Masse

  25 shared

• Keira E. Mahoney

  Yale University

  24 shared

Labs

• Malaker LabPI

Awards & honors

• 2026 Camille Dreyfus Teacher-Scholar
• 2026 David Y. Gin Award
• 2025-26 Prize Teaching Fellow
• 2025 International Glycoconjugate Organization Hakomuri Youn…
• 2024 Lloyd J. Old STAR