A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format.

Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by ß-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.

Total plasma N-glycomic signature of SARS-CoV-2 infection.

Total plasma protein N-glycosylation (TPNG) changes are a hallmark of many diseases. Here, we analyzed the TPNG of 169 COVID-19 patients and 12 healthy controls, using mass spectrometry, resulting in the relative quantification of 85 N-glycans. We found a COVID-19 N-glycomic signature, with 59 glycans differing between patients and controls, many of them additionally differentiating between severe and mild COVID-19. Tri- and tetra-antennary N-glycans were increased in patients, showing additionally elevated levels of antennary α2,6-sialylation. Conversely, bisection of di-antennary, core-fucosylated, nonsialylated glycans was low in COVID-19, particularly in severe cases, potentially driven by the previously observed low levels of bisection on antibodies of severely diseased COVID-19 patients. These glycomic changes point toward systemic changes in the blood glycoproteome, particularly involvement of acute-phase proteins, immunoglobulins and the complement cascade. Further research is needed to dissect glycosylation changes in a protein- and site-specific way to obtain specific functional leads.

Deciphering disease through glycan codes: leveraging lectin microarrays for clinical insights.

Glycosylation, a crucial posttranslational modification, plays a significant role in numerous physiological and pathological processes. Lectin microarrays, which leverage the high specificity of lectins for sugar binding, are ideally suited for profiling the glycan spectra of diverse and complex biological samples. In this review, we explore the evolution of lectin detection technologies, as well as the applications and challenges of lectin microarrays in analyzing the glycome profiles of various clinical samples, including serum, saliva, tissues, sperm, and urine. This review not only emphasizes significant advancements in the high-throughput analysis of polysaccharides but also provides insight into the potential of lectin microarrays for diagnosing and managing diseases such as tumors, autoimmune diseases, and chronic inflammation. We aim to provide a clear, concise, and comprehensive overview of the use of lectin microarrays in clinical settings, thereby assisting researchers in conducting clinical studies in glycobiology.

Comprehensive Glycomic and Glycoproteomic Analyses of Human Programmed Cell Death Protein 1 Extracellular Domain.

Human programmed cell death protein 1 (hPD-1) is an essential receptor in the immune checkpoint pathway. It has played an important role in cancer therapy. However, not all patients respond positively to the PD-1 antibody treatment, and the underlying mechanism remains unknown. PD-1 is a transmembrane glycoprotein, and its extracellular domain (ECD) is reported to be responsible for interactions and signal transduction. This domain contains 4 N-glycosylation sites and 25 potential O-glycosylation sites, which implicates the importance of glycosylation. The structure of hPD-1 has been intensively studied, but the glycosylation of this protein, especially the glycan on each glycosylation site, has not been comprehensively illustrated. In this study, hPD-1 ECD expressed by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells was analyzed; not only N- and O-glycosylation sites but also the glycans on these sites were comprehensively analyzed using mass spectrometry. In addition, hPD-1 ECD binding to different anti-hPD-1 antibodies was tested, and N-glycans were found functioned differently. All of this glycan information will be beneficial for future PD-1 studies.

Clinical glycoprotein mass spectrometry: The future of disease detection and monitoring.

Protein glycosylation is the co- and/or post-translational modification of proteins with oligosaccharides (glycans). This process is not template based and can introduce a heterogeneous set of glycan modifications onto substrate proteins. Glycan structures preserve biomolecular information from the cell, with glycoproteins from different cell types and tissues displaying distinct patterns of glycosylation. Several decades of research have revealed that glycan structures also differ between normal physiology and disease. This suggests that the information stored in glycoproteins and glycans can be utilized for disease diagnosis and monitoring. Methods that enable sensitive and site-specific measurement of protein glycosylation in clinical settings, such as nano-flow liquid chromatography tandem mass spectrometry, are therefore essential. The purpose of this perspective is to discuss recent advances in mass spectrometry and the potential of these advances to facilitate the detection and monitoring of disease-specific glycoprotein glycoforms. Glycoproteomics, the system-wide characterization of glycoprotein identity inclusive of site-specific characterization of carbohydrate modifications on proteins, and glycomics, the characterization of glycan structures, will be discussed in this context. Quantitative measurement of glycopeptide markers via parallel reaction monitoring is highlighted. The development of promising glycopeptide markers for autoimmune disease, liver disease, and liver cancer is discussed. Synthetic glycopeptide standards, ambient ionization mass spectrometry, and consideration of glyco-biomarkers in two- and three-dimensional space within tissue will be critical to the advancement of this field. The authors envision a future in which glycoprotein mass spectrometry workflows will be integrated into clinical settings, to aid in the rapid diagnosis and monitoring of disease.

12-Plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tag-Enabled High-Throughput Quantitative Glycomics.

Glycans, which are ubiquitously distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as molecular recognition and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probe labeling strategies has greatly advanced analysis of glycans. However, the requirement of high-throughput and robust quantitative analysis still calls for the development of more advanced tools. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the mass-defect strategy and expanded the multiplexing capacity to 12 channels without changing the chemical structure of the SUGAR tag, achieving a threefold enhancement in throughput compared with the original design and managing to perform high-throughput N-glycan analysis in a single LC - MS/MS injection. Herein, we present detailed methods for the synthesis of 12-plex SUGAR isobaric tags, the procedure to release and label the N-glycans from proteins, and the analysis by high-resolution LC-MS/MS, as well as data processing to achieve multiplexed quantitative glycomics.

[Applications of chromatography in glycomics].

Glycomics, an emerging "omics" technology that was developed after genomics and proteomics, is a discipline that studies the composition, structure, and functions of glycomes in cells, tissues, and organisms. Glycomics plays key roles in understanding the laws of major life activities, disease prevention and treatment, and drug quality control and development. At present, the structural analysis of glycans relies mainly on mass spectrometry. However, glycans have low abundance in biological samples. In addition, factors such as variable monosaccharide compositions, differences in glycosidic bond positions and modes, diverse branching structures, contribute to the complexity of the compositions and structures of glycans, posing great challenges to glycomics research. Liquid chromatography can effectively remove matrix interferences and enhance glycan separation to improve the mass spectrometric response of glycans. Thus, liquid chromatography and liquid chromatography coupled with mass spectrometry are important technical tools that have been actively applied to solve these problems; these technologies play indispensable roles in glycomics research. Different studies have highlighted similarities and differences in the applications of various types of liquid chromatography, which also reflects the versatility and flexibility of this technology. In this review, we first discuss the enrichment methods for glycans and their applications in glycomics research from the perspective of chromatographic separation mechanisms. We then compare the advantages and disadvantages of these methods. Some glycan-enrichment modes include affinity, hydrophilic interactions, size exclusion, and porous graphitized carbon adsorption. A number of newly developed materials exhibit excellent glycan-enrichment ability. We enumerate the separation mechanisms of reversed-phase high performance liquid chromatography (RP-HPLC), high performance anion-exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC), and porous graphitic carbon (PGC) chromatography in the separation and analysis of glycans, and describe the applications of these methods in the separation of glycans, glycoconjugates, and glyco-derivatives. Among these methods, HILIC and PGC chromatography are the most widely used, whereas HPAEC and RP-HPLC are less commonly used. The HILIC and RP-HPLC modes are often used for the separation of derived glycans. The ionization efficiency and detectability of glycans are significantly improved after derivatization. However, the derivatization process is relatively cumbersome, and byproducts inevitably affect the accuracy and completeness of the detection results. HPAEC and PGC chromatography exhibit good separation effects on nonderivative glycans, but issues related to the detection integrity of low-abundance glycans owing to their poor detection effect continue to persist. Therefore, the appropriate analytical method for a specific sample or target analyte or mutual verification must be selected. Finally, we highlight the research progress in various chromatographic methods coupled with mass spectrometry for glycomics analysis. Significant progress has been made in glycomics research in recent years owing to advancements in the development of chromatographic separation techniques. However, several significant challenges remain. As the development of novel separation materials and methods continues, chromatographic techniques may be expected to play a critical role in future glycomics research.

Integration of Web-Based Tools to Visualize, Integrate, and Interpret Glycogene Expression and Glycomics Data.

Glycosylation is the most abundant and diverse post-translational modification occurring on proteins. Glycans play important roles in modulating cell adhesion, growth, development, and differentiation. Changes in glycosylation affect protein structure and function and contribute to disease processes. Therefore, understanding glycosylation patterns is key for the identification of targets for the diagnosis of diseases, cellular states, and therapy. Glycosylation is a non template-driven process governed by the action of numerous enzymes and substrate availability that varies among cell types and species. Therefore, qualitative and quantitative assessment of global glycosylation and individual glycans remains challenging because it requires integration of multiple complex data types. Glycan structure and quantity data are often integrated with assessments of gene expression to aid contextualization of observed glycosylation changes within biological processes. However, correlating glycogene expression to the glycan structure is challenging because transcriptional changes may not always concur with the final gene product; there is often a lack of information on nucleotide sugar pools, and the final glycan structure is the result of many different glycogenes acting in concert. To overcome these challenges, interactive online tools are emerging as key resources for facilitating the analysis and integration of glycomics and glycogene expression data. Importantly, these tools work in concurrence with glycan biosynthetic schemes and therefore provide a clear indication of the molecular pathways where the glycan and glycogene are involved. In this chapter, we describe the applications of four freely available online tools that can be used for integrated visualization, interpretation, and presentation of RNAseq and glycomics results.

Modulation of AAV9 Galactose Binding Yields Novel Gene Therapy Vectors and Predicts Cross-Species Differences in Glycan Avidity.

Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extrahepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect in vivo AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a 6-fold reduction in liver RNA expression and a 10-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extrahepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies.

EBV-positive glycoproteins associated with nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is closely related to Epstein-Barr virus (EBV) infection, and glycosylation of proteins is associated with precancerous lesions and carcinogenesis of NPC, and viral glycoproteins mediates the fusion of viruses with B cells or epithelial cells in the infection stage, promoting the conversion of normal epithelial cells into cancer cells. In the process of occurrence and development of NPC, various glycoproteins in the body promote or inhibit the proliferation, invasion, metastasis, and drug resistance of tumor cells, such as the tumor inhibitory effect of NGX6 and inhibin B (INHBB); the cancer-promoting effect of tenascin-C (TNC), fibronectin 1 (FN1), insulin-like growth factor binding protein-3 (IGFBP3), serglycin, and its core protein; and some effects of glycosylation of immune proteins on immunotherapy in NPC. This article provides an overview of the research progress on the interaction of glycoproteins associated with EBV infection with the occurrence and development of NPC.

Automated high throughput IgG N-glycosylation sample preparation method development on the Tecan Freedom EVO platform.

Introduction: Glycomics, focusing on the role of glycans in biological processes, particularly their influence on the folding, stability and receptor interactions of glycoconjugates like antibodies, is vital for our understanding of biology. Changes in immunoglobulin G (IgG) N-glycosylation have been associated with various physiological and pathophysiological conditions. Nevertheless, time-consuming manual sample preparation is one of the limitations in the glycomics diagnostic implementation. The study aimed to develop an automated method for sample preparation on the Tecan Freedom Evo 200 platform and compare its efficiency and precision with the manual counterpart. Materials and methods: The initial method development included 32 pooled blood plasma technical replicates. An additional 24 pooled samples were used in the method comparison along with 78 random duplicates of plasma samples collected from 10,001 Dalmatians biobank to compare the manual and automated methods. Results: The development resulted in a new automated method. For the automated method, glycan peaks comprising 91% of the total sample glycan showed a variation of less than 5% while 92% of the total sample showed a variation of less than 5% for the manual method. The results of the Passing-Bablok regression indicated no differences between the automated and manual methods for 12 glycan peaks (GPs). However, for 8 GPs systematic difference was present, while both systematic and proportional differences were present for four GPs. Conclusions: The developed automated sample preparation method for IgG glycan analysis reduced exposure to hazardous chemicals and offered a simplified workflow. Despite slight differences between the methods, the new automated method showed high precision and proved to be highly comparable to its manual counterpart.

Multi Omics Applications in Biological Systems.

Traditional methodologies often fall short in addressing the complexity of biological systems. In this regard, system biology omics have brought invaluable tools for conducting comprehensive analysis. Current sequencing capabilities have revolutionized genetics and genomics studies, as well as the characterization of transcriptional profiling and dynamics of several species and sample types. Biological systems experience complex biochemical processes involving thousands of molecules. These processes occur at different levels that can be studied using mass spectrometry-based (MS-based) analysis, enabling high-throughput proteomics, glycoproteomics, glycomics, metabolomics, and lipidomics analysis. Here, we present the most up-to-date techniques utilized in the completion of omics analysis. Additionally, we include some interesting examples of the applicability of multi omics to a variety of biological systems.

Elevated level of multibranched complex glycan reveals an allergic tolerance status.

BACKGROUND: Allergen immunotherapy (AIT) is the only disease-modifying therapy that can achieve immune tolerance in patients through long-term allergen stimulation. Glycans play crucial roles in allergic disease, but no information on changes in glycosylation related to an allergic tolerance status has been reported. METHODS: Fifty-seven patients with house dust mite (HDM) allergies were enrolled. Twenty-eight patients were not treated with AIT, 19 patients had just entered the AIT maintenance treatment phase, and 10 patients had been in the AIT maintenance phase for more than 1 year. Serum protein N-glycans were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), which included linkage-specific sialylation information. RESULTS: Eighty-four N-glycans were identified in all three groups. Compared with the patients treated without AIT, the patients treated with AIT for a shorter time showed downregulated expression of high-mannose glycans and upregulated expression of α2,6 sialic acid. The patients treated with AIT in the maintenance phase for over 1 year, which was considered the start of immunological tolerance, showed downregulated expression of biantennary N-glycans and upregulated expression of multibranched and complex N-glycans. Nine N-glycans were changed between allergic and allergic-tolerant patients. CONCLUSIONS: The glycan form changed from mannose to a more complex type as treatment time increased, and multibranched complex glycans have the potential to be used as a monitoring indicator of immune tolerance. This serum N-glycome analysis provided important information for a deeper understanding of AIT treatment at the molecular level.

Recent advances in N-glycan biomarker discovery among human diseases.

N-glycans play important roles in a variety of biological processes. In recent years, analytical technologies with high resolution and sensitivity have advanced exponentially, enabling analysts to investigate N-glycomic changes in different states. Specific glycan and glycosylation signatures have been identified in multiple diseases, including cancer, autoimmune diseases, nervous system disorders, and metabolic and cardiovascular diseases. These glycans demonstrate comparable or superior indicating capability in disease diagnosis and prognosis over routine biomarkers. Moreover, synchronous glycan alterations concurrent with disease initiation and progression provide novel insights into pathogenetic mechanisms and potential treatment targets. This review elucidates the biological significance of N-glycans, compares the existing glycomic technologies, and delineates the clinical performance of N-glycans across a range of diseases.

Mass spectrometry imaging of N-linked glycans: Fundamentals and recent advances.

With implications in several medical conditions, N-linked glycosylation is one of the most important posttranslation modifications present in all living organisms. Due to their nontemplate synthesis, glycan structures are extraordinarily complex and require multiple analytical techniques for complete structural elucidation. Mass spectrometry is the most common way to investigate N-linked glycans; however, with techniques such as liquid-chromatography mass spectrometry, there is complete loss of spatial information. Mass spectrometry imaging is a transformative analytical technique that can visualize the spatial distribution of ions within a biological sample and has been shown to be a powerful tool to investigate N-linked glycosylation. This review covers the fundamentals of mass spectrometry imaging and N-linked glycosylation and highlights important findings of recent key studies aimed at expanding and improving the glycomics imaging field.

Comprehensive Glycomic and Proteomic Analysis of Mouse Striatum and Lateral Hypothalamus Following Repeated Exposures to Cocaine or Methamphetamine.

Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry-based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.

Apolipoprotein-CIII O-Glycosylation Is Associated with Micro- and Macrovascular Complications of Type 2 Diabetes.

Apolipoprotein-CIII (apo-CIII) inhibits the clearance of triglycerides from circulation and is associated with an increased risk of diabetes complications. It exists in four main proteoforms: O-glycosylated variants containing either zero, one, or two sialic acids and a non-glycosylated variant. O-glycosylation may affect the metabolic functions of apo-CIII. We investigated the associations of apo-CIII glycosylation in blood plasma, measured by mass spectrometry of the intact protein, and genetic variants with micro- and macrovascular complications (retinopathy, nephropathy, neuropathy, cardiovascular disease) of type 2 diabetes in a DiaGene study (n = 1571) and the Hoorn DCS cohort (n = 5409). Mono-sialylated apolipoprotein-CIII (apo-CIII1) was associated with a reduced risk of retinopathy (ß = -7.215, 95% CI -11.137 to -3.294) whereas disialylated apolipoprotein-CIII (apo-CIII2) was associated with an increased risk (ß = 5.309, 95% CI 2.279 to 8.339). A variant of the GALNT2-gene (rs4846913), previously linked to lower apo-CIII0a, was associated with a decreased prevalence of retinopathy (OR = 0.739, 95% CI 0.575 to 0.951). Higher apo-CIII1 levels were associated with neuropathy (ß = 7.706, 95% CI 2.317 to 13.095) and lower apo-CIII0a with macrovascular complications (ß = -9.195, 95% CI -15.847 to -2.543). In conclusion, apo-CIII glycosylation was associated with the prevalence of micro- and macrovascular complications of diabetes. Moreover, a variant in the GALNT2-gene was associated with apo-CIII glycosylation and retinopathy, suggesting a causal effect. The findings facilitate a molecular understanding of the pathophysiology of diabetes complications and warrant consideration of apo-CIII glycosylation as a potential target in the prevention of diabetes complications.

Editors' Choice-Perspective-Deciphering the Glycan Kryptos by Solid-State Nanopore Single-Molecule Sensing: A Call for Integrated Advancements Across Glyco- and Nanopore Science.

Glycans, or complex carbohydrates, are information-rich biopolymers critical to many biological processes and with considerable importance in pharmaceutical therapeutics. Our understanding, though, is limited compared to other biomolecules such as DNA and proteins. The greater complexity of glycan structure and the limitations of conventional chemical analysis methods hinder glycan studies. Auspiciously, nanopore single-molecule sensors-commercially available for DNA sequencing-hold great promise as a tool for enabling and advancing glycan analysis. We focus on two key areas to advance nanopore glycan characterization: molecular surface coatings to enhance nanopore performance including by molecular recognition, and high-quality glycan chemical standards for training.

Streamlining the Analysis of Proteins from Snake Venom.

Investigating snake venom is necessary for developing new treatments for envenoming and harnessing the therapeutic potential that lies within venom toxins. Despite considerable efforts in previous research, several technical challenges remain for characterizing the individual components within such complex mixtures. Here, we present native and top-down mass spectrometry (MS) workflows that enable the analysis of individual venom proteins within complex mixtures and showcase the utility of these methodologies on King cobra (Ophiophagus hannah) venom. First, we coupled ion mobility spectrometry for separation and electron capture dissociation for charge reduction to resolve highly convoluted mass spectra containing multiple proteins with masses ranging from 55 to 127 kDa. Next, we performed a top-down glycomic analysis of a 25.5 kDa toxin, showing that this protein contains a fucosylated complex glycan. Finally, temperature-controlled nanoelectrospray mass spectrometry facilitated the top-down sequence analysis of a ß-cardiotoxin, which cannot be fragmented by collisional energy due to its disulfide bond pattern. The work presented here demonstrates the applicability of new and promising MS methods for snake venom analysis.

The effects of immortalization on the N-glycome and proteome of CDK4-transformed lung cancer cells.

Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g. CDK4) or enable telomere lengthening. To demonstrate the changes in phenotype upon CDK4-transformation, we performed LC-MS/MS glycomic and proteomic characterizations of a human lung cancer primary cell line (DTW75) and a CDK4-transformed cell line (GL01) derived from DTW75. We observed that the primary and CDK4-transformed cells expressed significantly different levels of sialylated, fucosylated, and sialofucosylated N-glycans. Specifically, the primary cells expressed higher levels of hybrid- and complex-type sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex-type fucosylated and sialofucosylated N-glycans. Further, we compared the proteomic differences between the cell lines and found that CDK4-transformed cells expressed higher levels of RNA-binding and adhesion proteins. Further, we observed that the CDK4-transformed cells changed N-glycosylation after 31 days in cell culture, with a decrease in high-mannose and increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identifying these changes between primary and CDK4-transformed cells will provide useful insight when adapting cell lines that more closely resemble in vivo physiological conditions.