Abundant Human Proteins Research Articles

Phosphorylation Code of Human Nucleophosmin Includes Four Cryptic Sites for Hierarchical Binding of 14-3-3 Proteins

Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in the nucleolus but can also shuttle to the cytosol. NPM1 is regulated by multisite phosphorylation, yet molecular consequences of site-specific NPM1 phosphorylation remain elusive. Here we identify four 14-3-3 protein binding sites in NPM1 concealed within its oligomerization and α-helical C-terminal domains that are found phosphorylated in vivo. By combining mutagenesis, in-cell phosphorylation and PermaPhos technology for site-directed incorporation of a non-hydrolyzable phosphoserine mimic, we show how phosphorylation promotes NPM1 monomerization and partial unfolding, to recruit 14-3-3 dimers with low-micromolar affinity. Using fluorescence anisotropy we quantified pairwise interactions of all seven human 14-3-3 isoforms with four recombinant NPM1 phosphopeptides and assessed their druggability by fusicoccin. This revealed a complex hierarchy of 14-3-3 affinities toward the primary (S48, S293) and secondary (S106, S260) sites, differentially modulated by the small molecule. As three of these 14-3-3 binding phosphosites in NPM1 reside within signal sequences, this work suggests a mechanism of NPM1 regulation by which NPM1 phosphorylation can promote 14-3-3 binding to affect NPM1 shuttling between cell compartments. It also provides further evidence that phosphorylation-induced structural rearrangements of globular proteins serve to expose otherwise cryptic 14-3-3-binding sites that are important for cellular function.

Potential of Collagen as an Active Ingredient in Cosmetics

Skin is the part of the outer body that is most often exposed to external factors, especially ultraviolet radiation. The skin has an important role in supporting the appearance so that it can affect human social life. Skin aging is a process of decreasing the body's physiological functions that cannot be avoided. The aging process of the skin can be caused by a decrease in collagen density and replacement of essential structural proteins so that the skin loses its integrity and elasticity. Collagen is the most abundant protein in humans which can be found in bones, muscles, skin and tendons. Several studies have shown that damaged collagen fibers can be replaced by new fibers when collagen production is stimulated, which is why many cosmetic industries are competing to develop the use of collagen. Collagen has shown potential as an antioxidant, skin moisturizer, and anti-aging. Therefore, this article examines the benefits of collagen, especially in the cosmetic field. This article also discusses the sources of collagen with the best properties as active ingredients for skin care products

A novel mutation in collagen transport protein, MIA3 gene, detected in a patient with clinical symptoms of Ehlers-Danlos hypermobile syndrome.

Collagen, the most abundant human protein, is a significant component of the extracellular matrix (ECM) in tissues and organs like skin, bone, ligaments, and tendons. Collagen secretion is a complex, multistage process involving many molecules. A protein playing one of the main functions in this process is TANGO1 encoded by MIA3 gene. In the hypermobile type of Ehlers-Danlos syndrome (hEDS), one of the most common collagenopathies with no known genetic background, disrupted secretion of many molecules (including collagen) was observed. The aim of this study was the evaluation of the MIA3 gene role in hEDS patients. One hundred patients with clinically diagnosed hEDS and negative next-generation sequencing (NGS) testing for connective tissue disorder (e.g. Ehlers-Danlos syndrome, osteogenesis imperfect (OI), Marfan syndrome, and others) were tested for molecular changes in the MIA3 gene. Among the 100 tested patients, 14 single structural changes in the MIA3 gene were detected. Thirteen were missense benign or likely benign, while 1 variant (c.567dup, p.Leu1880ThrfsTer6) was truncating the TANGO1 protein. We suppose that the presence of truncating variant (c.5637dup) in the MIA3 gene and disrupted secretion of connective tissue protein may be one of the pathogenic mechanisms of clinical symptoms present in the tested patient, but these findings require a more comprehensive multidimensional investigation.

Cancer cell survival depends on collagen uptake into tumor-associated stroma

Collagen I, the most abundant protein in humans, is ubiquitous in solid tumors where it provides a rich source of exploitable metabolic fuel for cancer cells. While tumor cells were unable to exploit collagen directly, here we show they can usurp metabolic byproducts of collagen-consuming tumor-associated stroma. Using genetically engineered mouse models, we discovered that solid tumor growth depends upon collagen binding and uptake mediated by the TEM8/ANTXR1 cell surface protein in tumor-associated stroma. Tumor-associated stromal cells processed collagen into glutamine, which was then released and internalized by cancer cells. Under chronic nutrient starvation, a condition driven by the high metabolic demand of tumors, cancer cells exploited glutamine to survive, an effect that could be reversed by blocking collagen uptake with TEM8 neutralizing antibodies. These studies reveal that cancer cells exploit collagen-consuming stromal cells for survival, exposing an important vulnerability across solid tumors with implications for developing improved anticancer therapy.

Conformational Analysis of Fluoro-, Chloro-, and Proteo-Alkene Gly-Pro and Pro-Pro Isosteres to Mimic Collagen.

Collagen is the most abundant human protein, with the canonical sequence (Gly-Pro-Hyp)n in its triple helix region. Cis-trans isomerization of the Xaa-Pro amide has made two of these amide bonds the target of alkene replacement: the Gly-Pro and the Pro-Hyp positions. The conformations of Gly-Pro and Pro-Pro (as a Pro-Hyp model) fluoro-, chloro-, and proteo-alkene mimic models were investigated computationally to determine whether these alkenes can stabilize the polyproline type II (PPII) conformation of collagen. Second-order Møller-Plesset (MP2) calculations with various basis sets were used to perform the conformational analyses and locate stationary points. The calculation results predict that fluoro- and chloro-alkene mimics of Gly-Pro and Pro-Pro can participate in n→π* donation to stabilize PPII conformations, yet they are poor n→π* acceptors, shifting the global minima away from PPII conformations. For the proteo-alkene mimics, the lack of significant n→π* interactions and unstable PPII-like geometries explains their known destabilization of the triple helix in collagen-like peptides.

Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms

The seven 14-3-3 isoforms are highly abundant human proteins encoded by similar yet distinct genes. 14-3-3 proteins recognize phosphorylated motifs within numerous human and viral proteins. Here, we analyze by X-ray crystallography, fluorescence polarization, mutagenesis and fusicoccin-mediated modulation the structural basis and druggability of 14-3-3 binding to four E6 oncoproteins of tumorigenic human papillomaviruses. 14-3-3 isoforms bind variant and mutated phospho-motifs of E6 and unrelated protein RSK1 with different affinities, albeit following an ordered affinity ranking with conserved relative KD ratios. Remarkably, 14-3-3 isoforms obey the same hierarchy when binding to most of their established targets, as supported by literature and a recent human complexome map. This knowledge allows predicting proportions of 14-3-3 isoforms engaged with phosphoproteins in various tissues. Notwithstanding their individual functions, cellular concentrations of 14-3-3 may be collectively adjusted to buffer the strongest phosphorylation outbursts, explaining their expression variations in different tissues and tumors.

Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

Mapping the Minimal Promoter Sequence for Human Prolidase (Peptidase D; PEPD‐ gene)

Collagen, the most abundant protein in humans, is the primary component of the extracellular matrix. Collagen is also a critical reservoir of proline that, along with hydroxyproline, comprise one‐fourth of the amino acid composition of collagen. During collagen turnover, sequential action of matrix metalloproteinases and peptidases convert collagen into dipeptides. In the subsequent final rate‐limiting step, prolidase (PEPD) hydrolyzes dipeptides containing C‐terminal proline or hydroxyproline into the constituent amino acids, which are then recycled towards collagen synthesis. Due to its critical role in collagen turnover and protein metabolism, prolidase is a key player in several physiological and pathological processes, including wound healing, cell proliferation, inflammation, and carcinogenesis. Consequently, alterations in prolidase expression can profoundly affect normal physiological processes and cause pathological changes. The goal of this study is to narrow down such knowledge gaps, especially, in our understanding of the regulation of PEPD transcription. First, towards determining a functionally active promoter region of PEPD (PEPDp), we cloned a 1587 base pair (bp) DNA region—spanning 1537 bp upstream, and 50 bp downstream, of the transcription start site (TSS)—of PEPD gene into a firefly luciferase reporter plasmid. We confirmed that the PEPDp is functionally active by demonstrating its ability to robustly drive the luciferase expression in the HEK293T cell line. Second, we sought to determine the minimal region of PEPDp that is sufficient and necessary to drive gene expression. We subcloned two progressively shorter deletion fragments of PEPDp—537 bp and 387 bp long into the firefly luciferase reporter system and then compared their promoter activity with that of the full‐length PEPDp. Our findings thus indicate that the lack of any cancellation in the promoter activity of the PEPDp deletion fragments points out that the functionally active region of PEPDp spans ~350 bp upstream and 50 bp downstream of the transcriptional start site (TSS), thereby advancing our knowledge of the molecular mechanisms regulating prolidase expression.Support or Funding InformationThis work is partly supported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).

Cyclic Peptide Mimetic of Damaged Collagen.

Collagen is the most abundant protein in humans and the major component of human skin. Collagen mimetic peptides (CMPs) can anneal to damaged collagen in vitro and in vivo. A duplex of CMPs was envisioned as a macromolecular mimic for damaged collagen. The duplex was synthesized on a solid support from the amino groups of a lysine residue and by using olefin metathesis to link the N termini. The resulting cyclic peptide, which is a monomer in solution, binds to CMPs to form a triple helix. Among these, CMPs that are engineered to avoid the formation of homotrimers but preorganized to adopt the conformation of a collagen strand exhibit enhanced association. Thus, this cyclic peptide enables the assessment of CMPs for utility in annealing to damaged collagen. Such CMPs have potential use in the diagnosis and treatment of fibrotic diseases and wounds.

Molecular mechanics of Staphylococcus aureus adhesin, CNA, and the inhibition of bacterial adhesion by stretching collagen.

Bacterial adhesion to collagen, the most abundant protein in humans, is a critical step in the initiation and persistence of numerous bacterial infections. In this study, we explore the collagen binding mechanism of the multi-modular cell wall anchored collagen adhesin (CNA) in Staphylococcus aureus and examine how applied mechanical forces can modulate adhesion ability. The common structural-functional elements and domain organization of CNA are present across over 50 genera of bacteria. Through the use of molecular dynamics models and normal mode analysis, we shed light on the CNA’s structural and conformational dynamics and its interactions with collagen that lead to collagen binding. Our results suggest that the linker region, CNA165-173, acts as a hinge exhibiting bending, extensional, and torsional modes of structural flexibility and its residues are key in the interaction of the CNA-collagen complex. Steered molecular dynamics simulations were conducted with umbrella sampling. During the course of these simulations, the ‘locking’ latch from the CNA N2 domain was dissociated from its groove in the CNA N1 domain, implying the importance of the latch for effective ligand binding. Finally, we observed that the binding efficiency of the CNA N1-N2 domains to collagen decreases greatly with increasing tensile force application to the collagen peptides. Thus, CNA and similar adhesins might preferentially bind to sites in which collagen fibers are cleaved, such as in wounded, injured, or inflamed tissues, or in which the collagenous tissue is less mature. As alternative techniques for control of bacterial infection are in-demand due to the rise of bacterial antibiotic resistance, results from our computational studies with respect to the mechanoregulation of the collagen binding site may inspire new therapeutics and engineering solutions by mechanically preventing colonization and/or further pathogenesis.

Metals and immunity

Metal‐sequestering innate immune proteins participate in the host/pathogen interaction. The S100 protein family includes calprotectin (S100A8/S100A9), psoriasin (S100A7), and S100A12, and these proteins exhibit antimicrobial activity that results from their ability to chelate transition metals in the extracellular space and thereby reduce the availability of essential metal nutrients to pathogens. Calprotectin is a versatile metal‐sequestering protein that can capture a number of divalent first‐row transition metals at an unusual hexahistidine site, whereas psoriasin and S100A12 can selectively deplete zinc. Recent biochemical and biophysical studies that address the coordination chemistry and host‐defense function of these abundant human proteins will be presented.Support or Funding InformationThis research was supported by the NSF (CHE‐1352132), the Camille & Henry Dreyfus Foundation, and the MIT Center for Environmental Health Sciences (NIH Grant P30‐ES002109).

Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface.

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in NS than in HIS, as validated by immunofluorescence.

Development of an enhanced metaproteomic approach for deepening the microbiome characterization of the human infant gut.

The establishment of early life microbiota in the human infant gut is highly variable and plays a crucial role in host nutrient availability/uptake and maturation of immunity. Although high-performance mass spectrometry (MS)-based metaproteomics is a powerful method for the functional characterization of complex microbial communities, the acquisition of comprehensive metaproteomic information in human fecal samples is inhibited by the presence of abundant human proteins. To alleviate this restriction, we have designed a novel metaproteomic strategy based on double filtering (DF) the raw samples, a method that fractionates microbial from human cells to enhance microbial protein identification and characterization in complex fecal samples from healthy premature infants. This method dramatically improved the overall depth of infant gut proteome measurement, with an increase in the number of identified low-abundance proteins and a greater than 2-fold improvement in microbial protein identification and quantification. This enhancement of proteome measurement depth enabled a more extensive microbiome comparison between infants by not only increasing the confidence of identified microbial functional categories but also revealing previously undetected categories.

Single-Molecule Tracking of Collagenase on Native Type I Collagen Fibrils Reveals Degradation Mechanism

Collagen, the most abundant human protein, is the principal component of the extracellular matrix and plays important roles in maintaining tissue and organ integrity. Highly resistant to proteolysis, fibrillar collagen is degraded by specific matrix metalloproteases (MMPs). Degradation of fibrillar collagen underlies processes including tissue remodeling, wound healing, and cancer metastasis. However, the mechanism of native collagen fibril degradation remains poorly understood. Here we present the results of high-resolution tracking of individual MMPs degrading type I collagen fibrils. MMP1 exhibits cleavage-dependent biased and hindered diffusion but spends 90% ± 3% of the time in one of at least two distinct pause states. One class of exponentially distributed pauses (class I pauses) occurs randomly along the fibril, whereas a second class of pauses (class II pauses) exhibits multistep escape kinetics and occurs periodically at intervals of 1.3 ± 0.2 μm and 1.5 ± 0.2 μm along the fibril. After these class II pauses, MMP1 moved faster and farther in one direction along the fibril, indicative of biased motion associated with cleavage. Simulations indicate that 5% ± 2% of the class II pauses result in the initiation of processive collagen degradation, which continues for bursts of 15 ± 4 consecutive cleavage events. These findings provide a mechanistic paradigm for type I collagen degradation by MMP1 and establish a general approach to investigate MMP-fibrillar collagen interactions. More generally, this work demonstrates the fundamental role of enzyme-substrate interactions including binding and motion in determining the activity of an enzyme on an extended substrate.

Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D(2)O and compare with experimental observations.

Single Molecule Study of the Motion of Matrix Metalloprotease MMP1 on Type I Collagen Fiber Shows Proteolysis Driven Hindered Biased Diffusion

Diffusion plays an important role in many biological processes. Using single molecule fluorescence techniques, we have studied the diffusive motion of matrix metalloprotease MMP1 on Type I collagen fiber from rat tail. Collagen is the most abundant protein in humans. Fibril forming Type I collagen is the main component of the extracellular matrix (ECM) that supports and defines most tissues. Degradation of collagen in the ECM by matrix metalloproteases (MMPs) is an important process in tissue remodeling.

Binding of Matrix Metalloproteinase Inhibitors to Extracellular Matrix: 3D‐QSAR Analysis

Binding to the extracellular matrix, one of the most abundant human protein complexes, significantly affects drug disposition. Specifically, the interactions with extracellular matrix determine the free concentrations of small molecules acting in tissues, including signaling peptides, inhibitors of tissue remodeling enzymes such as matrix metalloproteinases, and other drug candidates. The nature of extracellular matrix binding was elucidated for 63 matrix metalloproteinase inhibitors, for which the association constants to an extracellular matrix mimic were reported here. The data did not correlate with lipophilicity as a common determinant of structure-nonspecific, orientation-averaged binding. A hypothetical structure of the binding site of the solidified extracellular matrix surrogate was analyzed using the Comparative Molecular Field Analysis, which needed to be applied in our multi-mode variant. This fact indicates that the compounds bind to extracellular matrix in multiple modes, which cannot be considered as completely orientation-averaged and exhibit structural dependence. The novel comparative molecular field analysis models, exhibiting satisfactory descriptive and predictive abilities, are suitable for prediction of the extracellular matrix binding for the untested chemicals, which are within applicability domains. The results contribute to a better prediction of the pharmacokinetic parameters such as the distribution volume and the tissue-blood partition coefficients, in addition to a more imminent benefit for the development of more effective matrix metalloproteinase inhibitors.

Type I Collagen and Collagen Mimetics as Angiogenesis Promoting Superpolymers

Angiogenesis, the development of blood vessels from the pre-existing vasculature, is a key component of embryogenesis and tissue regeneration. Angiogenesis also drives pathologies such as tumor growth and metastasis, and hemangioma development in newborns. On the other hand, promotion of angiogenesis is needed in tissues with vascular insufficiencies, and in bioengineering, to endow tissue substitutes with appropriate microvasculatures. Therefore, much research has focused on defining mechanisms of angiogenesis, and identifying pro- and anti-angiogenic molecules. Type I collagen, the most abundant protein in humans, potently stimulates angiogenesis in vitro and in vivo. Crucial to its angiogenic activity appears to be ligation and possibly clustering of endothelial cell (EC) surface alpha 1 beta 1/alpha 2 beta 1 integrin receptors by the GFPGER(502-507) sequence of the collagen fibril. However, additional aspects of collagen structure and function that may modulate its angiogenic properties are discussed. Moreover, type I collagen and fibrin, another angiogenic polymer, share several structural features. These observations suggest strategies for creating "angiogenic superpolymers", including: modifying type I collagen to influence its biological half-life, immunogenicity, and integrin binding capacity; genetically engineering fibrillar collagens to include additional integrin binding sites or angiogenic determinants, and remove unnecessary or deleterious sequences without compromising fibril integrity; and exploring the suitability of poly(ortho ester), PEG-lysine copolymer, tubulin, and cholesteric cuticle as collagen mimetics, and suggesting means of modifying them to display ideal angiogenic properties. The collagenous and collagen mimetic angiogenic superpolymers described here may someday prove useful for many applications in tissue engineering and human medicine.

Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen.

Type I collagen is the most abundant protein in humans, and it helps to maintain the integrity of many tissues via its interactions with cell surfaces, other extracellular matrix molecules, and growth and differentiation factors. Nearly 50 molecules have been found to interact with type I collagen, and for about half of them, binding sites on this collagen have been elucidated. In addition, over 300 mutations in type I collagen associated with human connective tissue disorders have been described. However, the spatial relationships between the known ligand-binding sites and mutation positions have not been examined. To this end, here we have created a map of type I collagen that includes all of its ligand-binding sites and mutations. The map reveals the existence of several hot spots for ligand interactions on type I collagen and that most of the binding sites locate to its C-terminal half. Moreover, on the collagen fibril some potentially relevant relationships between binding sites were observed including the following: fibronectin- and certain integrin-binding regions are near neighbors, which may mechanistically relate to fibronectin-dependent cell-collagen attachment; proteoglycan binding may potentially impact upon collagen fibrillogenesis, cell-collagen attachment, and collagen glycation seen in diabetes and aging; and mutations associated with osteogenesis imperfecta and other disorders show apparently nonrandom distribution patterns within both the monomer and fibril, implying that mutation positions correlate with disease phenotype. These and other observations presented here may provide novel insights into evaluating type I collagen functions and the relationships between its binding partners and mutations.

Separation of human serum albumin components by RP-HPLC and CZE and their characterization by ESI-MS

Human serum albumin (HSA) is one of the most abundant human proteins and has been shown to be heterogeneous. A RP-HPLC method has been developed to separate HSA components in commercially available preparations. Separations were carried out on Aquapore RP-300, C8 columns using gradient elution with a combination of acetonitrile/water mobile phases containing 0.05% trifluoroacetic acid as ion-pairing agent. Optimum resolution was attained on narrow-bore columns using a stepwise, linear gradient that incorporated a shallow intermediate step of 0.20%/min in Mobile Phase B. Under similar elution conditions, separations carried out on standard-size columns showed the expected decrease in resolution due to increased peak widths. A comparative analysis of three commercial products highlighted qualitative and quantitative differences. Capillary zone electrophoresis was used for the analysis of collected RP-HPLC fractions. Results indicated that while the HPLC separation was incomplete, one of the major HPLC peaks was primarily composed of one of the three main components typically separated by CZE. ESI-MS was used to characterize the two major RP-HPLC fractions and also showed that the HPLC separation was incomplete. The MaxEnt transform of the HPLC peaks was consistent with components all being HSA and closely related derivatives.