Analysis of oligosaccharides derived from heparin by ion-pair reversed-phase chromatography/mass spectrometry.

Current chromatographic and mass spectrometric techniques have limitations for analyzing heparin and heparin oligomers due to their high polarity, structural diversity, and sulfate lability. A rapid method for the analysis of heparin oligosaccharides was developed using ion-pair reversed-phase ultraperformance liquid chromatography coupled with electrospray quadruple time-of-flight mass spectrometry (IPRP-UPLC ESI Q-TOF MS). The method utilizes an optimized buffer system containing a linear pentylamine and a unique additive, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), to achieve highly efficient separation together with enhanced mass response of heparin oligosaccharides. Analyses of a heparin oligosaccharide test mixture, dp6 through dp22, reveal that the chromatographic conditions enable baseline resolution of isomeric heparin oligosaccharides (dp6) and produce intact molecular ions with no sulfate losses during mass spectrometric analysis. In addition, the described conditions are amenable to the detection of heparin oligosaccharides in positive ion mode, yield stronger positive ion signals for corresponding oligosaccharides compared to the negative ion mode, and allow identification of structural isomers by an MS/MS approach. Because sensitive detection of oligosaccharides is also achieved with ultraviolet (UV) detection, the method utilizes a dual detection scheme (UV and MS in series) along with IPRP UPLC to simultaneously obtain quantification (UV) and characterization (MS) data for heparin oligosaccharides. The broad potential of this new method is further demonstrated for the analysis of a low-molecular-weight heparin (LMWH) preparation from porcine heparin. This approach will be of particular utility for profiling the molecular entities of heparin materials, as well as for structural variability comparison for samples from various sources.

Characterization of protein impurities and site-specific modifications using peptide mapping with liquid chromatography and data independent acquisition mass spectrometry.

Liquid chromatography (LC)-based peptide mapping is extensively used for establishing protein identity, assessing purity, and detecting post-translational modifications (PTMs) of recombinant proteins in the biopharmaceutical industry. However, current LC-UV/MS peptide mapping methods require multiple analyses and MS/MS experiments to identify protein contaminants and site-specific PTMs. This manuscript evaluated an alternative approach for protein characterization via peptide mapping employing a data independent LC-MS acquisition strategy with an alternate low and elevated collision energy scanning. The acquired peptide precursor and fragment information was utilized for effective identification of peptide sequences and site-specific modifications within a single LC run. The peptide MS signal intensities were reliably measured and used to estimate relative concentrations of PTMs and/or proteins contaminating the target protein. The method was evaluated using tryptic digests of yeast enolase and alcohol dehydrogenase. LC-eluted peptides were successfully sequenced and covered 97% target protein sequences. Protein impurities and site-specific modifications (e.g., M-oxidation and N-deamidation) were identified and quantified.

Comparison of 1-D and 2-D LC MS/MS methods for proteomic analysis of human serum.

1-D and 2-D LC methods were utilized for proteome analysis of undepleted human serum. Separation of peptides in 2-D LC was performed either with strong cation exchange (SCX)-RP chromatography or with an RP-RP 2-D LC approach. Peptides were identified by MS/MS using a data-independent acquisition approach. A peptide retention prediction model was used to highlight the potential false-positive peptide identifications. When applying selected data filtration, we identified 52 proteins based on 316 peptides in serum in 1-D LC setup. One hundred and eighty-four proteins/1036 peptides and 142 proteins/905 peptides were identified in RP-RP and SCX-RP 2-D LC, respectively. The performance of both 2-D LC methods for proteomic analysis is critically compared.

Phosphopeptide enrichment using microscale titanium dioxide solid phase extraction.

Identification of phosphopeptides by MS is challenging due to their relatively low abundance in proteomic samples and their limited ionization efficiency. Various affinity enrichment methods have been used in the literature. Titanium dioxide SPE devices have been recently proposed as an alternative to immobilized metal affinity chromatography for phosphopeptide enrichment. This study evaluates the TiO(2 )method using sorbent packed in a 96 well microscale extraction plate operated using a vacuum manifold. The phosphopeptide recovery and enrichment selectivity were investigated at various loading conditions. The effectiveness of organic additives such as dihydroxybenzoic acid derivatives and other nonaliphatic carboxylic acids on enrichment selectivity was examined. The performance of TiO(2) was compared to IMAC sorbent. The results suggest that various additives improve the enrichment selectivity by effectively interfering with the acidic peptides binding to TiO(2) sorbent. Interaction of phosphopeptides with sorbent is also affected, which leads to overall reduction in phosphopeptide recovery. The new SPE device was successfully utilized for the extraction of phosphopeptides from yeast lysate digest using 2,5-dihydroxybenzoic acid to minimize the interference from nonphosphorylated peptides.

Comparative profiling of human saliva by intact protein LC/ESI-TOF mass spectrometry.

Human saliva is finding increasing interest for proteomic and biomarker-discovery studies, due to the ease of collection and potential for simpler processing workflows compared to serum or plasma. However, it is known that salivary protein composition can vary with physiological and environmental factors. In this work, we have examined intra- and inter-person variability of saliva protein composition using an LC/MS methodology to profile low molecular weight human salivary proteins. Whole saliva was analyzed from four individuals over three consecutive days. Additional samples were used to determine baseline analytical and sample processing variation and to identify phosphoproteins. Individuals were observed to have a similar salivary protein pattern over multiple days, although the expression levels of particular proteins were variable. Significant differences in protein profiles were observed between subjects, allowing for delineation of individuals based on their protein profile. Comparison with alkaline phosphatase treated saliva revealed that several identified proteins were singly, doubly, or triply phosphorylated.

Mixed-mode chromatography for fractionation of peptides, phosphopeptides, and sialylated glycopeptides.

A mixed-mode chromatographic (MMC) sorbent was prepared by functionalizing the silica sorbent with a pentafluorophenyl (PFP) ligand. The resulting stationary phase provided a reversed-phase (RP) retention mode along with a relatively mild strong cation-exchange (SCX) retention interaction. While the mechanism of interaction is not entirely clear, it is believed that the silanols in the vicinity of the perfluorinated ligand act as strongly acidic sites. The 2.1 mm x 150 mm column packed with such sorbent was applied to the separation of peptides. Linear RP gradients in combination with salt steps were used for pseudo two-dimensional (2D) separation and fractionation of tryptic peptides. An alternative approach of using linear cation-exchange gradients combined with RP step gradients was also investigated. Besides the attractive forces, the ionic repulsion contributed to the retention mechanism. The analytes with strong negatively charged sites (phosphorylated peptides, sialylated glycopeptides) eluted in significantly different patterns than generic tryptic peptides. This retention mechanism was employed for the isolation of phosphopeptides or sialylated glycopeptides from non-functionalized peptide mixtures. The mixed-mode column was utilized in conjunction with a phosphopeptide enrichment solid phase extraction (SPE) device packed with metal oxide affinity chromatography (MOAC) sorbent. The combination of MOAC and mixed-mode chromatography (MMC) provided for an enhanced extraction selectivity of phosphopeptides and sialylated glycopeptides peptides from complex samples, such as yeast and human serum tryptic digests.

Effects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separations.

The effects of the column length, the particle size, the gradient length and the flow rate of a nanoLC system on peptide peak capacity were investigated and compared. Columns packed with 1.7 microm and 3 microm C(18) materials into pieces of 75 microm capillary tubing of various lengths were tested with different gradient lengths and flow rates. While increasing the length of a column packed with the 1.7 microm material helped improve peptide peak capacity at the whole range of the tested gradient lengths (24-432 min), little improvement in peak capacity was observed with the increase of the length of a column packed with the 3 microm material unless a gradient longer than 50 min was carried out. Up to 30% of peak capacity increase was observed when a column's length is doubled, with little reduction in the throughput. In most cases, more than 50% of the increase in peak capacity was obtained with the reduction in the particle size from 3 microm to 1.7 microm. With the same backpressure generated, a shorter 1.7-microm-particle column outperformed a longer column packed with the 3 microm material. In a flow rate range of 100-700 nl/min, increasing the flow rate improved peak capacity for columns packed with 1.7 microm and 3 microm materials.

Development of an online two-dimensional nano-scale liquid chromatography/mass spectrometry method for improved chromatographic performance and hydrophobic peptide recovery.

An online two-dimensional (2D) strong cation-exchange (SCX)/reversed-phase (RP) nano-scale liquid chromatography/mass spectrometry (nanoLC/MS) method was developed for improved separation and hydrophobic peptide recovery. Sharper and more symmetric RP peaks were observed with the use of a "band re-focusing method", in which an analytical RP column with more hydrophobicity than the RP trap column was used in the system. To recover hydrophobic peptides still unreleased from the SCX column after a conventional salt step gradient due to hydrophobic interaction, a RP step gradient from 10% to 30% acetonitrile (ACN) was applied to the SCX column in the presence of a high salt concentration following the salt gradient. There were 301 unique hydrophobic E. coli peptides identified from the RP fractions. These peptides, which were 19% of all E. coli peptides identified from a 2D run, would not have been identified without the application of the RP gradient to the SCX column.

Evaluation of multidimensional (ion-exchange/reversed-phase) protein separations using linear and step gradients in the first dimension.

The performance characteristics of multidimensional liquid chromatographic protein separations were evaluated using on-line electrospray mass detection, and a novel workflow for automated LC/MS data processing. Two-dimensional ion exchange/reversed-phase LC separations of Escherichia coli cytosol were conducted using either a continuous linear or discontinuous step gradient in the first dimension. Chromatographic profiles of the top 100 most abundant components were characterized to assess overall separation reproducibility within each mode, and to characterize differences in component distribution between the two modes of operation. Analysis of the resulting data indicates that multidimensional separations of complex protein mixtures can be done reproducibly. Furthermore, under the conditions employed within this study, a linear first dimension gradient was more effective at fractionating the protein mixture, distributing fewer major components to multiple second dimension cycles than an equivalent step gradient. The application of on line mass spectrometry, and automated processing of the resulting data, proved valuable for producing component level analysis of multidimensional protein separations.

Complete sequencing of anti-vancomycin fab fragment by liquid chromatography-electrospray ion trap mass spectrometry with a combination of database searching and manual interpretation of the MS/MS spectra.

Sequencing of anti-vancomycin monoclonal antibody (mAb) Fab region (48,000 Da) was carried out using liquid chromatography-electrospray ionization ion trap mass spectrometry (LC/ESI-MS). Comprehensive strategies were employed to ensure complete sequence coverage. The sequence information was obtained from the spectra of collision-induced dissociation (CID) (MS/MS) of the protonated proteolytic peptides resulting from multiple enzymatic digestions of reduced/S-carboxymethylated (RCM) light chain and Fd fragment. Database searching of the spectra against the published immunoglobulin G (IgG) sequences allowed the identification of all the peptides in constant domains as well as partial sequences in variable domains. The rest of the sequences were deduced by manual interpretation of the peptide tandem mass spectrometry (MS/MS) spectra. The analysis showed that the N-terminus of the heavy chain was modified by the conversion of a glutamine residue to pyroglutamic acid.

Characterization of therapeutic oligonucleotides using liquid chromatography with on-line mass spectrometry detection.

A method for the analysis and characterization of therapeutic and diagnostic oligonucleotides has been developed using a combination of liquid chromatography and mass spectrometry (LC-MS). The optimized ion-pairing buffers permit a highly efficient separation of native and chemically modified antisense oligonucleotides (AS-ODNs) from their metabolites or failure synthetic products. The mobile phases were MS compatible, allowing for direct and sensitive analysis of components eluting from the column. The method was applied for the quantitation and characterization of AS-ODNs, including phosphorothioates and 2'-O-methyl-modified phosphorothioates. Tandem LC-MS analysis confirmed the identity of the oligonucleotide metabolites, failure products, the presence of protection groups not removed after synthesis, and the extent of depurination or phosphorothioate backbone oxidation.

Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography.

Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension.

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction.

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.

Purification of dye-labeled oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography.

Singly- and dually-labeled synthetic oligonucleotides were purified by ion-pair reversed-phase high-performance liquid chromatography using a 50x4.6-mm column packed with porous, 2.5 micrometer C(18) sorbent. We studied the mechanism of dye-labeled oligonucleotide retention in order to improve the quality of purification. By-products of oligonucleotide synthesis were characterized by liquid chromatography with mass spectrometry detection (LC-MS). We purified oligonucleotides labeled with 6-carboxyfluorescein (6FAM), hexachlorofluorescein (HEX), tetrachlorofluorescein (TET), carboxytetramethylrhodamine (TAMRA) and indodicarboxycyanine (Cy3) dyes, as well as dually-labeled TaqMan probes. Purification of a 0.1-micromole oligonucleotide synthesis in a single injection was demonstrated.

Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab.

Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied.

Determination of N-glycosylation sites and site heterogeneity in a monoclonal antibody by electrospray quadrupole ion-mobility time-of-flight mass spectrometry.

This paper presents an improved analytical method for glycosylation structural characterizations of a monoclonal antibody (mAb) using a newly developed quadrupole ion-mobility time-of-flight (ESI-Q-IM-TOF) mass spectrometer. Using this method, high-resolution mass spectra were acquired to produce the overall glycosylation profile of the mAb. Additionally, the light and heavy chains from the reduced antibody were separated in the gas phase by the ion mobility functionality of the instrument, allowing accurate mass measurement of each subunit. Furthermore, the glycan sequences, as well as the glycosylation site, were determined by a two-step sequential fragmentation process using the unique dual-collision-cell design of the instrument, thus providing detailed characterizations of the glycan structures.

Peptide retention prediction applied to proteomic data analysis.

A retention prediction model was developed for peptides separated in reversed-phase chromatography. The model was utilized to identify and exclude the false positive (FP) peptide identifications obtained via database search. The selected database included human proteins, as well as decoy sequences of random proteins. The FP peptide detection rate was defined either as number of retention time outliers, or random decoy sequence identifications. The FP rate for various MASCOT scores was calculated. The peptides identified in one-dimensional (1D) and two-dimensional (2D) liquid chromatography/mass spectrometry (LC/MS) experiments were validated by prediction models. Multi-dimensional LC was based on two orthogonal reversed-phase chromatography modes; prediction models were successfully applied for data filtering in both separation dimensions.

Use of an integrated MS--multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies.

Liquid chromatography/mass spectrometry (LC/MS) peptide maps have become a basic tool for characterizing proteins of biological and pharmaceutical interest. The ability to generate reproducible maps with high protein sequence coverage is a central goal of methods development. We have applied a recently developed analytical approach (termed LC/MS(E)) to LC/MS peptide mapping. Using the LC/MS(E) approach, the mass detector alternates between a low-energy scanning mode (MS) for accurate mass peptide precursor identification, and an elevated-energy mode (MS(E)) for generation of accurate mass multiplex peptide fragmentation data. In this paper, we evaluate this analytical approach against a tryptic digest of yeast enolase. From the low-energy data, high peptide map coverage (98% of sequence from peptides >3 amino acids) was reproducibly obtained. The MS signal for essentially equimolar peptides varied over 2 orders of magnitude in intensity, and peptide intensities could be precisely and reproducibly measured. Using the temporal constraint that MS(E) peptide fragment ions exhibit chromatographic profiles that parallel the precursor ions that generated them, we were able to produce accurate mass time-resolved MS/MS information for all enolase peptides with sufficient abundance to produce a detectable fragment ion.

A method to reduce gradient delay time of NanoLC.

A novel method to dramatically reduce the delay time of a nanoLC gradient is described. The gradient is divided into two parts, and each part is formed at different flow rates. The beginning part is formed and delivered to the inlet of the column at a higher-than-normal flow rate. With the formation of the rest of the gradient at a normal flow rate, the whole gradient is further delivered through the column at the same normal flow rate. To form the gradient with the desired slope, the volumetric gradient slope was kept constant, independent of the flow rate. A gradient delay time reduction of 12.5-16 min was observed with the reported method. The resulting gradient profiles and chromatograms were very similar to those obtained with a conventional method. Comparable retention time reproducibility was observed between the two methods.

Identification of N-linked glycosylation sites using glycoprotein digestion with pronase prior to MALDI tandem time-of-flight mass spectrometry.

Glycopeptides are typically prepared by cleaving the proteins with specific proteolytic enzymes, such as trypsin. The resulting glycopeptides tend to have weak mass spectrometry ion signals (ESI or MALDI) due to their relatively large molecular weight. The identification of glycosylation sites with tandem mass spectrometry is further complicated by fragmentation of both the peptide backbone and the glycan moiety. We explored a method using a nonspecific enzyme, pronase, to generate small glycopeptides (between two and six amino acids). These glycopeptides were enriched and desalted using a microscale hydrophilic interaction chromatography extraction device prior to MALDI QTof MS analysis. MALDI matrix, 2, 5-dihydroxybenzoic acid, doped with ammonium triscitrate, was utilized for analysis. Sodiated ions were observed as minor ions, while protonated ions were enhanced dramatically with this matrix. Collision-induced dissociation was performed on both the protonated and sodiated ions. MS/MS fragmentation spectra reveal that proton has greater affinity for the peptide moiety, while the sodium cation tends to associate with the sugar moiety. Characteristic fragment patterns allowed for identifications of glycosylation sites for both the protonated and the sodiated precursor ions. Model proteins, horseradish peroxidase and alpha1-acid glycoproteins, were analyzed to illustrate the identification of N-linked glycosylation sites and data interpretation algorithm.