Barry Boyes

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples: II. Selectivity effects observed in the separation of several peptide and protein mixtures

Changes in band spacing as a function of temperature and/or gradient steepness were investigated for four peptide or protein samples. Reversed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthetic peptide mixture and a storage protein sample from wheat were studied. Simultaneous changes in gradient steepness and temperature were found to provide considerable control over band spacing and sample resolution. The effects of temperature and gradient steepness on selectivity in these systems appear to be complementary. Simultaneous optimization of both temperature and gradient steepness thus represents a powerful and convenient means of controlling band spacing and separation. Because of the complexity of these sample chromatograms, computer simulation proved to be a useful tool in both interpreting these experiments and in optimizing final separations.

Fast high performance liquid chromatography separations for proteomic applications using Fused-Core® silica particles.

The separation range of superficially porous particles (Fused-Core®) has been extended by design of particles with 160 Å pores. These particles show superior kinetics (lower resistance to mass transfer), allowing fast separations of peptides and small proteins (molecular weights of <15,000). The high efficiency and relatively low back pressure of these 2.7 μm Fused-Core particles has been maintained so that separations can be performed with conventional HPLC instruments. Longer columns can be used for higher resolution of complex mixtures of peptides, such as proteolytic digests. Highly reproducible separations of peptides at elevated temperatures with low pH mobile phases are maintained as a result of a stable bonded stationary phase. The utility of such highly stable materials is exemplified by separations of problematic amyloid peptides at low pH (TFA mobile phase) at an operational temperature of 100 °C. To address the issue of poor peptide peak shape in formic acid-containing mobile phases we show that the addition of 10-20 mM ammonium formate improves peak shape, retention and load tolerance of peptides. Use of the Fused-Core particle materials for separations of synthetic peptides and tryptic digests yields peak capacities that are comparable to those obtained using columns packed with sub-2-μm particles, but with less than one-half of the operating back pressure. A peak capacity of 530 was obtained in 150 min on coupled columns of HALO Peptide ES-C18 with a combined length of 250 mm.

Liquid chromatography-selected reaction monitoring (LC-SRM) approach for the separation and quantitation of sialylated N-glycans linkage isomers.

The study of N-linked glycans is among the most challenging bioanalytical tasks because of their complexity and variety. The presence of glycoform families that differ only in branching and/or linkage position makes the identification and quantitation of individual glycans exceedingly difficult. Quantitation of these individual glycans is important because changes in the abundance of these isomers are often associated with significant biomedical events. For instance, previous studies have shown that the ratio of α2-3 to α2-6 linked sialic acid (SA) plays an important role in cancer biology. Consequently, quantitative methods to detect alterations in the ratios of glycans based on their SA linkages could serve as a diagnostic tool in oncology, yet traditional glycomic profiling cannot readily differentiate between these linkage isomers. Here, we present a liquid chromatography-selected reaction monitoring (LC-SRM) approach that we demonstrate is capable of quantitating the individual SA linkage isomers. The LC method is capable of separating sialylated N-glycan isomers differing in α2-3 and α2-6 linkages using a novel superficially porous particle (Fused-Core) Penta-HILIC (hydrophilic interaction liquid chromatography) column. SRM detection provides the relative quantitation of each SA linkage isomer, and minimizes interferences from coeluting glycans that are problematic for UV/Fluorescence based quantitation. With our approach, the relative quantitation of each SA linkage isomer is obtained from a straightforward liquid chromatography-mass spectrometry (LC-MS) experiment.

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.

Selectivity optimization of reversed-phase high-performance liquid chromatographic peptide and protein separations by varying bonded-phase functionality

Several chemical bonded-phase modified silicas were prepared using sterically protected monofunctional silane reagents which varied widely in structure and polarity. Since some of these bonded-phase packing materials are highly polar (hydrophilic), resistance to acid-catalyzed bonded-phase loss by hydrolysis was examined, and observed to remain high even for the highly polar Diol bonded-phase functionality. Modification of the surface of 300 A pore size, fully hydroxylated and base-deactivated silica microspheres with these sterically protected silanes yielded HPLC column packing materials for examination of separation selectivities in reversed-phase separations of peptide and protein mixtures. Distinct separation selectivities were apparent for each bonded-phase functionality. Selectivity differences ranged from limited band spacing changes for steric-protected C18 and C8 bonded-phases, to reversal of elution order for the more polar C3 and CN bonded phases. The use of column-based selectivity differences between sequential reversed-phase separation steps is used for the two-step HPLC isolation of a recombinant human amyloid precursor polypeptide fragment from a crude bacterial extract.

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC‐MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS‐based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC‐MS analysis of permethylated N‐glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments.

Optimization of data-dependent acquisition parameters for coupling high-speed separations with LC-MS/MS for protein identifications.

Recent developments in chromatography, such as ultra-HPLC and superficially porous particles, offer significantly improved peptide separation. The narrow peak widths, often only several seconds, can permit a 15-min liquid chromatography run to have a similar peak capacity as a 60-min run using traditional HPLC approaches. In theory, these larger peak capacities should provide higher protein coverage and/or more protein identifications when incorporated into a proteomic workflow. We initially observed a decrease in protein coverage when implementing these faster chromatographic approaches, due to data-dependent acquisition (DDA) settings that were not properly set to match the narrow peak widths resulting from newly implemented, fast separation techniques. Oversampling of high-intensity peptides lead to low protein-sequence coverage, and tandem mass spectra (MS/MS) from lower-intensity peptides were of poor quality, as automated MS/MS events were occurring late on chromatographic peaks. These observations led us to optimize DDA settings to use these fast separations. Optimized DDA settings were applied to the analysis of Trypanosome brucei peptides, yielding peptide identifications at a rate almost five times faster than previously used methodologies. The described approach significantly improves protein identification workflows that use typical available instrumentation.

The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS)

AbstractPeptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein’s function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications. Graphical Abstractᅟ

Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC).

The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in α2-3 and α2-6 linkages, while these glycans remain attached to peptides.

Peptide retention prediction using hydrophilic interaction liquid chromatography coupled to mass spectrometry

A model that predicts retention for peptides using a HALO® penta-HILIC column and gradient elution was created. Coefficients for each amino acid were derived using linear regression analysis and these coefficients can be summed to predict the retention of peptides. This model has a high correlation between experimental and predicted retention times (0.946), which is on par with previous RP and HILIC models. External validation of the model was performed using a set of H. pylori samples on the same LC-MS system used to create the model, and the deviation from actual to predicted times was low. Apart from amino acid composition, length and location of amino acid residues on a peptide were examined and two site-specific corrections for hydrophobic residues at the N-terminus as well as hydrophobic residues one spot over from the N-terminus were created.