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Interferons are signaling proteins that belong to the large class of cytokines and human interferons which are classified based on the type of receptor interactions: type I, II and III. IFNα2b belongs to the type I interferon class with a major therapeutic application for the treatment of hepatitis B and C infections. A recombinant form of IFNα2b expressed in E. coli, known as IntronA, has been approved by US Food and Drug Administration (FDA). IFN γ, also known as type II interferon, plays a significant role in the inhibition of viral replication. Actimmune® is a US Food and Drug Administration (FDA) approved version of IFN γ for the indication of reducing infections associated with chronic granulomatous disease and severe malignant osteopetrosis. In this study we have applied advanced analytical methods for the characterization of IFNα2b and IFN γ produced from Pichia pastoris. The multi-enzyme digestion approach has been developed to allow measurement of 100% sequence coverage and detailed analysis of post-translational variants and degradation products. In this manner, we identified the following variants in IFN α2b: N-terminal residual leader sequence, an amino acid substitution, oxidation of methionine residues and two sites of high mannose N-glycosylation. In the Pichia IFN γ produced material, our approach detected variants resulting from glycosylation, C-terminal proteolysis, oxidation of methionine residues and deamidation. In this manner, the analytical program was able to support rapid process development as well as identify product variants and degradation products in the resulting product.

With the advent of biosimilars to the U.S. market, it is important to have better analytical tools to ensure product quality from batch to batch. In addition, the recent popularity of using a continuous process for production of biopharmaceuticals, the traditional bottom-up method, alone for product characterization and quality analysis is no longer sufficient. Bottom-up method requires large amounts of material for analysis and is labor-intensive and time-consuming. Additionally, in this analysis, digestion of the protein with enzymes such as trypsin could induce artifacts and modifications which would increase the complexity of the analysis. On the other hand, a top-down method requires a minimum amount of sample and allows for analysis of the intact protein mass and sequence generated from fragmentation within the instrument. However, fragmentation usually occurs at the N-terminal and C-terminal ends of the protein with less internal fragmentation. Herein, we combine the use of the complementary techniques, a top-down and bottom-up method, for the characterization of human growth hormone degradation products. Notably, our approach required small amounts of sample, which is a requirement due to the sample constraints of small scale manufacturing. Using this approach, we were able to characterize various protein variants, including post-translational modifications such as oxidation and deamidation, residual leader sequence, and proteolytic cleavage. Thus, we were able to highlight the complementarity of top-down and bottom-up approaches, which achieved the characterization of a wide range of product variants in samples of human growth hormone secreted from Pichia pastoris.

Influenza hemagglutinin is a surface glycoprotein related to virus invasion and host immune system response. Understanding site specific glycosylation of hemagglutinin will increase our knowledge about virus evolution and can improve the design and quality of vaccines. In our study, we used glycoproteomic analysis based on multienzyme digestion followed by LC tandem MS analysis to determine the glycosylation of Influenza hemagglutinin (H1/A/California/04/2009) using the following steps: PNGaseF treatment combined with trypsin or pepsin digestion was used to determine the glycosites and glycan occupancy. Three enzymes, trypsin, AspN, and pepsin, were used separately to generate suitable glycopeptides for online LC tandem MS analysis. The glycan structure of a given glycopeptide was determined by collision-induced dissociation MS/MS fragmentation, and the peptide backbone information was provided by collision-induced dissociation (CID)-MS3 fragmentation. With this approach, 100% sequence coverage of the hemagglutinin sample was obtained. Six glycosylation sites fitting the sequon N-X-S/T were successfully confirmed, and the glycan heterogeneity as well as the ratios of glycoforms were determined at each site.

We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional analysis (RNA sequencing). Two unusual types of glycan structures were observed in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the observed expression of the corresponding α-N-acetylneuraminide α-2,8-sialyltransferase 2 (ST8SiA2) and α-N-acetylneuraminide α-2,8-sialyltransferase 4 (ST8SiA4), by quantitative real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by analysis with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the search for novel glycan structures present at specific sites in sEGFR as well as other secreted oncoproteins such as Erbb2 as markers of disease progression in blood samples from cancer patients.

We report progress assembling the parts list for chromosome 17 and illustrate the various processes that we have developed to integrate available data from diverse genomic and proteomic knowledge bases. As primary resources, we have used GPMDB, neXtProt, PeptideAtlas, Human Protein Atlas (HPA), and GeneCards. All sites share the common resource of Ensembl for the genome modeling information. We have defined the chromosome 17 parts list with the following information: 1169 protein-coding genes, the numbers of proteins confidently identified by various experimental approaches as documented in GPMDB, neXtProt, PeptideAtlas, and HPA, examples of typical data sets obtained by RNASeq and proteomic studies of epithelial derived tumor cell lines (disease proteome) and a normal proteome (peripheral mononuclear cells), reported evidence of post-translational modifications, and examples of alternative splice variants (ASVs). We have constructed a list of the 59 "missing" proteins as well as 201 proteins that have inconclusive mass spectrometric (MS) identifications. In this report we have defined a process to establish a baseline for the incorporation of new evidence on protein identification and characterization as well as related information from transcriptome analyses. This initial list of "missing" proteins that will guide the selection of appropriate samples for discovery studies as well as antibody reagents. Also we have illustrated the significant diversity of protein variants (including post-translational modifications, PTMs) using regions on chromosome 17 that contain important oncogenes. We emphasize the need for mandated deposition of proteomics data in public databases, the further development of improved PTM, ASV, and single nucleotide variant (SNV) databases, and the construction of Web sites that can integrate and regularly update such information. In addition, we describe the distribution of both clustered and scattered sets of protein families on the chromosome. Since chromosome 17 is rich in cancer-associated genes, we have focused the clustering of cancer-associated genes in such genomic regions and have used the ERBB2 amplicon as an example of the value of a proteogenomic approach in which one integrates transcriptomic with proteomic information and captures evidence of coexpression through coordinated regulation.

In this study, we report a combined proteomic and peptidomic analysis of human plasma from patients with rheumatoid arthritis (RA) and controls. We used molecular weight cut-off filters (MWCO: 10kDa) to enrich low-molecular-weight (LMW) peptides from human plasma. The peptide fraction was analyzed without trypsin digestion by capillary reversed-phase high-performance liquid chromatography (HPLC) coupled to a linear ion trap-FT-MS system, which identified 771 unique peptides in the peptidome study (from 145 protein progenitors). An anti-albumin/anti-IgG column was used to remove albumin and immunoglobulin G (IgG) from the human plasma. After that, the albumin/IgG-depleted sample was fractionated into a bound fraction and an unbound fraction on a multi-lectin affinity column (M-LAC). LC-MS analysis of the corresponding tryptic digests identified 308 proteins using the M-LAC approach. Relative differences in the following protein classifications were observed in the RA human plasma samples compared with controls: structural proteins, immuno-related proteins, protease inhibitors, coagulation proteins, transport proteins and apolipoproteins. While some of these proteins/peptides have been previously reported to be associated with RA disease such as calgranulin A, B, C and C-reactive protein, several others were newly identified (such as thymosin beta4, actin, tubulin, and vimentin), which may further the understanding of the disease pathogenesis. Moreover, we have found that the peptidomic and glycoproteomic approaches were complementary and allow a more complete picture of the human plasma proteome which can be valuable in studies of disease etiology.

We have performed a detailed characterization of recombinant human growth hormone that included the identification of the entire sequence with disulfide linkages as well as subtle modifications by a sensitive liquid chromatography coupled online with tandem mass spectrometry (LC-MS) approach using the accurate peptide mass (FTICR MS) and sequence assignment (MS/MS measurement). The extent of oxidation, deamidation, and chain cleavages were measured by the ratio of peak areas of the nonmodified peptide vs. the sum of peak area of the nonmodified and modified peptides in the same LC-MS analysis. The subtle but distinct differences were found in the recombinant human growth from the three manufacturers (the follow-on, counterfeit, and the original innovator products). In relative comparison, the follow-on product had the highest degree of oxidation at methionine residues, followed by the counterfeit product, and the original innovator product had the least amount of oxidation at all three sites with the similar oxidation order. In cases, the oxidation order was Met14 > Met125 > Met170. In contrast, the follow-on had the least amount of deamidation at aspargine (Asn149), and the counterfeit had the highest degree of deamidation at this site. For the chain cleavage, the follow-on product had the highest cleavage occurring at T 10 peptide (between Asn99 and Ser100), the counterfeit had the highest cleavage on T4 peptide, (between Glu30 and Phe31), and the original innovator product with the least amount of cleavages on both sites. These subtle but distinct differences are likely because of nonidentical manufacturing, formulation procedures, and storage conditions.

Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.

Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses.

In a recent report, we introduced Extended Range Proteomic Analysis (ERPA), an intermediate approach between top-down and bottom-up proteomics, for the comprehensive characterization at the trace level (fmol level) of large and complex proteins. In this study, we extended ERPA to determine quantitatively the temporal changes that occur in the tyrosine kinase receptor, epidermal growth factor receptor (EGFR), upon stimulation. Specifically A 431 cells were stimulated with epidermal growth factor after which EGFR was immunoprecipitated at stimulation times of 0, 0.5, 2, and 10 min as well as 4 h. High sequence coverage was obtained (96%), and methods were developed for label-free quantitation of phosphorylation and glycosylation. A total of 13 phosphorylation sites were identified, and the estimated stoichiometry was determined over the stimulation time points, including Thr(P) and Ser(P) sites in addition to Tyr(P) sites. A total of 10 extracellular domain N-glycan sites were also identified, and major glycoforms at each site were quantitated. No change in the extent of glycosylation with stimulation was observed as expected. Finally potential binding partners to EGFR were identified based on changes in the amount of protein pulled down with EGFR as a function of time of stimulation. Many of the 19 proteins identified are known binding partners of EGFR. This work demonstrates that comprehensive characterization provides a powerful tool to aid in the study of important therapeutic targets. The detailed molecular information will prove useful in future studies in tissue.

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53–Cys165 and Cys182–Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies. On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method. Copyright © 2004 John Wiley & Sons, Ltd.

The advantage of using the two-dimensional separation method was demonstrated by the separation of the complex tryptic peptides of recombinant human tissue plasminogen activator (rt-PA) glycoprotein. This method hybridizes two analytical methods where fractions containing glycopeptides are collected from reversed-phase high-performance liquid chromatography (RP-HPLC) and separated by capillary zone electrophoresis (CZE). CZE readily resolved carbohydrate structural variants, based on sialic acid content and branching, on the same peptide. Nonglycosylated peptides incidentally collected in the same RP-HPLC fraction were well resolved from the glycopeptides. This combination of RP-HPLC and CZE was able to uniquely resolve all of the peptides and glycopeptide variants in rt-PA. Confirmation of the specific peaks in the CZE was made by matrix-assisted laser-induced ionization time-of-flight mass spectrometry. The advantages and disadvantages of the existing methods for carbohydrate characterization in the biotech industry were compared. The advantage of this approach is to provide a simple extension of the existing tryptic digest protocols to include carbohydrate analysis to a detailed level of microheterogeneity.

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS3 (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the posttranslational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138 - Cys154, Cys143 - Cys150, Cys282 - Cys396, Cys470 - Cys482, Cys471 - Cys484, and Cys475 - Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (> 70%). A successful methodology has been developed which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.

SummaryFOXP3 is a key transcription factor for regulatory T cell function. We report the crystal structure of the FOXP3 coiled-coil domain, through which a loose or transient dimeric association is formed and modulated, accounting for the activity variations introduced by disease-causing mutations or posttranslational modifications. Structure-guided mutagenesis revealed that FOXP3 coiled-coil-mediated homodimerization is essential for Treg function in vitro and in vivo. In particular, we identified human FOXP3 K250 and K252 as key residues for the conformational change and stability of the FOXP3 dimer, which can be regulated by protein posttranslational modifications such as reversible lysine acetylation. These studies provide structural and mechanistic explanations for certain disease-causing mutations in the coiled-coil domain of FOXP3 that are commonly found in IPEX syndrome. Overall, the regulatory machinery involving homooligomerization, acetylation, and heteroassociation has been dissected, defining atomic insights into the biological and pathological characteristics of the FOXP3 complex.

The SUN (Sad1-UNC-84 homology) domain is conserved in a number of nuclear envelope proteins involved in nuclear migration, meiotic telomere tethering, and antiviral responses. The LINC (linker of nucleoskeleton and cytoskeleton) complex, formed by the SUN and the nesprin proteins at the nuclear envelope, serves as a mechanical linkage across the nuclear envelope. Here we report the crystal structure of the SUN2 protein SUN domain, which reveals a homotrimer. The SUN domain is sufficient to mediate binding to the KASH (Klarsicht, ANC-1, and Syne homology) domain of nesprin 2, and the regions involved in the interaction have been identified. Binding of the SUN domain to the KASH domain is abolished by deletion of a region important for trimerization or by point mutations associated with nuclear migration failure. We propose a model of the LINC complex, where the SUN and the KASH domains form a higher ordered oligomeric network in the nuclear envelope. These findings provide the structural basis for understanding the function and the regulation of the LINC complex.

In an attempt to develop high producing mammalian cell lines expressing glucagon-like-peptide-1-antibody fusion proteins (GLP-1), we have noted that the N-terminal GLP-1 portion of the fusion protein was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. The majority of the N-terminal clipped product appeared to be due to the removal of the entire biologically active peptide (30 amino acids) from the intact molecule. A number of parameters that influenced the degradative process were investigated. Additionally, protease inhibitors specific for each class of protease were tested. Results suggested that one or more serine-threonine class of protease(s) were involved in this process and inhibitors that are specific for this class of protease, including benzamidine hydrochloride could significantly inhibit the proteolytic degradation of the fusion proteins. Identification of the specific proteases involved in this process by shotgun proteomics methodology will pave the way for engineering the CHOK1SV cell line which will serve as a superior host for the production of future fusion protein products.

In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an overexpressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to ensure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an online LC-MS strategy combining collision-induced dissociation (CID-MS(2)), electron-transfer dissociation (ETD-MS(2)), and CID of an isolated product ion derived from ETD (MS(3)) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS(3) step. The online LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a therapeutic monoclonal antibody, and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS(3) step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance not only provided facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The online LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins.

This paper describes approaches to optimize the chromatographic performance for our recently developed LC-MS platform, extended range proteomic analysis (ERPA), for comprehensive protein characterization at the ultratrace level. Large digested peptide fragments up to 10 kDa (e.g., from lysyl endopeptidase digestion) with or without modifications were well separated with high resolution using narrow bore (20 and 50 microm I.D.) poly(styrene-divinylbenzene) (PS-DVB) monolithic columns constructed by in situ solution polymerization. Importantly, the macroporous structure of the monolithic columns facilitated mass transport of large peptides with improved recovery relative to small pore size reversed-phase packings. High sequence coverage (95%), including identification of phosphorylated and glycosylated particles was achieved for beta-casein and epidermal growth factor receptor (EGFR) at the 4 and 20 fmol levels per injection, respectively, using the 20 microm I.D. PS-DVB monolithic column. For peptides with greater ionization efficiency, the detection limit could be lowered to approximately 400 zmol. Typically, the separation system produced a peak capacity of approximately 200 for a 10 cm column. This paper demonstrates that narrow-bore monolithic columns are suitable for high sensitivity and high-resolution separation of large peptide fragments by LC-MS analysis.

The CD3ϵγ and CD3ϵδ heterodimers along with the CD3ζζ homodimer are the signaling components of the T cell receptor (TCR). These invariant dimers are non-covalently associated on the T cell plasma membrane with a clone-specific (i.e. clonotypic) αβ heterodimer that binds its cognate ligand, a complex between a particular antigenic peptide, and an MHC molecule (pMHC). These four TCR dimers exist in a 1:1:1:1 stoichiometry. At the junction between the extracellular and transmembrane domains of each mammalian CD3ϵ, CD3γ, and CD3δ subunit is a highly conserved CXXC motif previously found to be important for thymocyte and T cell activation. The redox state of each CXXC motif is presently unknown. Here we show using LC-MS and a biotin switch assay that these CXXC segments are constitutively oxidized on resting and activated T cells, consistent with their measured reduction potential. NMR chemical shift perturbation experiments comparing a native oxidized CD3δ CXXC-containing segment with that of a mutant SXXS-containing CD3δ segment in LPPG (1-palmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)) micelles show extensive chemical shift differences in residues within the membrane-proximal motif as well as throughout the transmembrane and cytoplasmic domains as a result of the elimination of the native disulfide. Likewise, direct comparison of the native CD3δ segment in oxidizing and reducing conditions reveals numerous spectral differences. The oxidized CXXC maintains the structure within the membrane-proximal stalk region as well as that of its contiguous transmembrane and cytoplasmic domain, inclusive of the ITAM (immunoreceptor tyrosine-based activation motif) involved in signaling. These results suggest that preservation of the CD3 CXXC oxidized state may be essential for TCR mechanotransduction.

Glycation of interferon-beta-1b and human serum albumin was identified in a lyophilized glucose formulation by a sensitive LC-MS approach. The extent of glycation was measured by a label-free quantitation strategy. The glycation sites were determined by the accurate mass (FTICR MS) with MS/MS measurements on the corresponding tryptic peptides. The extent of glycation was measured by the ratio of the peak intensity between the glycated and the average value for three non-glycated peptides in the same run. Residues lysine 18 of interferon-beta-1b, and lysine 51, lysine 233, and lysine 545 of human serum albumin were more prone to be glycated than other sites in this lyophilized glucose formulation. Residues of lysine 51 and lysine 233 but not lysine 545 of human serum albumin are highly accessible to solvent as found in a solution storage study by Lapolla et al. The extent of glycation of both proteins and the number of glycation sites of human serum albumin were increased with the storage time at 25 degrees C. In total, two glycation sites of interferon beta-1b and 17 glycation sites of human serum albumin were identified in the lyophilized glucose formulation with a storage time at 25 degrees C of 35 days. Among the 17 glycation sites, only lysine 525 of human serum albumin has been found in vivo in diabetic patients by Shaklai et al. As expected, there was no glycation found on both interferon-beta-1b and human serum albumin in the control samples (similar lyophilized formulation but using mannitol instead of glucose).

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MS n ) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.

We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (100 000) and accurate mass (2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.

The outer membrane protein B (OmpB) of the typhus group rickettsiae is an immunodominant antigen and has been shown to provide protection against typhus in animal models. Consequently, OmpB is currently being considered as a potential rickettsiae vaccine candidate to be used in humans. The OmpB from virulent strains are heavily methylated while the attenuated strains are hypomethylated. Western blot analysis of partially digested OmpB revealed that one of the reactive fragments was located at the N-terminus (fragment A, aa 33-272). Recently, we have over expressed, purified, and chemically methylated the recombinant fragment A from Rickettsia prowazekii (Ap). The methylated Ap was thoroughly characterized by LC/MS/MS on the ProteomeX workstation. The protein sequence of Ap with and without methylation was 87.7% and 100% identified, respectively. This high sequence coverage enabled us to determine the sites and extent of methylation on the lysine residues in Ap. All the lysine residues except the C-terminus lysine were either mono-, di- or tri-methylated. In addition, carbamylation on the N-terminus glycine was identified using a combination of denovo sequencing (DeNovoX) and the pattern recognition (SALSA) program with accurate mass measurement. We demonstrated that the use of peptide identification (SEQUEST) in combination with SALSA and denovo sequencing provided a useful means to characterize the sequence and posttranslational modifications of given proteins.

This study uses multiple enzyme digests to increase the sequence coverage of proteins identified by the shotgun sequencing approach to proteomic analysis. The enzymes used were trypsin, Lys-C, and Asp-N, which cleave at arginine and lysine residues, lysine, and aspartic acid residues, respectively. This approach was evaluated with the glycoprotein, tissue plasminogen activator, t-PA and gave enhanced sequence coverage, compared with a single enzymatic digest. The approach was then evaluated with a complex proteomic sample, namely plasma. It was found that trypsin and Lys-C were able to detect overlapping but distinct sets of proteins and a digital recombination of the data gave a significant increase in both the number of protein identifications as well as an increase in the number of peptides identified per protein (which improves the certainty of the assignment).

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. Whereas IGFBP-1 and IGFBP-2 are not glycosylated, IGFBP-3 and IGFBP-4 are N-glycosylated and IGFBP-5 and IGFBP-6 are O-glycosylated. In this study we identified the glycosylation of IGFBP-4 using a nanoflow LC/MS/MS techniques. Although N-linked oligosaccharides are structurally diverse, their variants are well reported in the literature. Based on the molecular weight of the possible oligosaccharide moieties, we identified five different glycosylation isoforms of the protein. Identified glycans were biantennary and differ in the number of sialic acid terminal residues and/or core modification with fucose.

Atrial natriuretic peptide (ANP) is a peptide with 25 amino acid residues (hANP 4-28) and one intra-chain disulfide bond. We used the size-exclusion chromatography (SEC), sodium dodecyl sulfate in polyacrylamide gel electrophoresis (SDS-PAGE), and reversed-phase liquid chromatography with electrospray mass spectrometry (LC-MS) methods to examine the freeze-dried products of ANP to determine the types, sizes, and amounts of the multimer formation in different stability protocols (by varying the conditions with lyophilization cycles, excipients, storage temperatures, and times). Under the non-annealing lyophilization cycle or lyophilization with high concentration of bulking agent (mannitol), multimer formation increased with increasing storage times. Two kinds of multimers were observed; the major portion is reducible and the minor portion is non-reducible. The reducible multimers are disulfide-linked multimers as determined by LC-MS. The non-reducible multimer was mainly a dimer, possibly linked by a covalent bond between the side chain of tyrosine in one molecule and the dehydroalanine intermediate in another molecule, based on the evidences of the mass of the non-reducible dimer along with the elution position in SEC, and the change of the UV spectrum in the aromatic region. The analysis of degradants suggests that the mechanism start from an beta-elimination of disulfide linkage to form a free thiolate ion (HS-) and a dehydroalanine-type peptide intermediate. The HS- then catalyzed ANP to form the disulfide-linked multimers. The dehydroalanine-type ANP intermediate then reacted with another ANP molecule to form a non-disulfide-linked dimer through reaction with the side chain of tyrosine. These results suggest that the source of multimer formation be initiated by phase transition (from amorphous to crystalline phase) either in the freeze-dried process or during storage. That phase change may induce a drastic change in pH and moisture to damage the peptide. The detailed mechanism and the kinetics of ANP multimerization are discussed. The formation of the multimers was diminished by using the thermal treatment (the annealing step) with a proper ratio of mannitol to ANP peptide in the lyophilization, and/or increase of the acetate buffer concentration in the formulation.