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Selected Reaction Monitoring (SRM) is a method of choice for accurate quantitation of low-abundance proteins in complex backgrounds. This strategy is, however, sensitive to interference from other components in the sample that have the same precursor and fragment masses as the monitored transitions. We present here an approach to detect interference by using the expected relative intensity of SRM transitions. We also designed an algorithm to automatically detect the linear range of calibration curves. These approaches were applied to the experimental data of Clinical Proteomic Tumor Analysis Consortium (CPTAC) Verification Work Group Study 7 and show that the corrected measurements provide more accurate quantitation than the uncorrected data.

Glycosylation is a critical attribute for development and manufacturing of therapeutic monoclonal antibodies (mAbs) in the pharmaceutical industry. Conventional antibody glycan analysis is usually achieved by the 2-aminobenzamide (2-AB) hydrophilic interaction liquid chromatography (HILIC) method following the release of glycans. Although this method produces satisfactory results, it has limited use for screening a large number of samples because it requires expensive reagents and takes several hours or even days for the sample preparation. A simple and rapid glycan analysis method was not available. To overcome these constraints, we developed and compared 2 ultrafast methods for antibody glycan analysis (UMAG) that involve the rapid generation and purification of glycopeptides in either organic solvent or aqueous buffer followed by label-free quantification using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Both methods quickly yield N-glycan profiles of test antibodies similar to those obtained by the 2-AB HILIC-HPLC method. In addition, the UMAG method performed in aqueous buffer has a shorter assay time of less than 15 min, and enables high throughput analysis in 96-well PCR plates with minimal sample handling. This method, the fastest, and simplest as reported thus far, has been evaluated for glycoprofiling of mAbs expressed under various cell culture conditions, as well as for the evaluation of antibody culture clones and various production batches. Importantly the method sensitively captured changes in glycoprofiles detected by traditional 2-AB HILIC-HPLC or HILIC-UPLC. The simplicity, high speed, and low cost of this method may facilitate basic research and process development for novel mAbs and biosimilar products.

A desorption electrospray ionization (DESI) source has been coupled to an ion mobility time-of-flight mass spectrometer for the analysis of proteins. Analysis of solid-phase horse heart cytochrome c and chicken egg white lysozyme proteins with different DESI solvents and conditions shows similar mass spectra and charge state distributions to those formed when using electrospray to analyze these proteins in solution. The ion mobility data show evidence for compact ion structures [when the surface is exposed to a spray that favors retention of "nativelike" structures (50:50 water:methanol)] or elongated structures [when the surface is exposed to a spray that favors "denatured" structures (49:49:2 water:methanol:acetic acid)]. The results suggest that the DESI experiment is somewhat gentler than ESI and under appropriate conditions, it is possible to preserve structural information throughout the DESI process. Mechanisms that are consistent with these results are discussed.

A field modulation approach for high-throughput ion mobility/time-of-flight analyses of complex mixtures has been developed using a split-field drift tube. In this approach, complex mixtures of peptides, such as those that arise from tryptic digestion of protein mixtures, are separated by nanocolumn liquid chromatography, ionized by electrospray ionization, and analyzed by ion mobility/ time-of-flight techniques. The split-field drift tube allows parent ions to be separated based on differences in their low-field mobilities through the first-field region before entering the second region. For increased throughput, the magnitude of the field in the second region can be modulated throughout an LC separation in order to favor transmission of different types of ions: parent ions at low fields; fragments from primarily [M+3H] 3+ peptides at moderate fields; or, fragmentation of [M+3H] 3+ and [M+2H] 2+ species at higher fields. We demonstrate the approach with two examples: a mixture of tryptic peptides from digestion of hemoglobin; and a complex mixture of tryptic peptides from digestion of human plasma.

The present work seeks to illuminate the underlying principles which control the aggregation of chiral building blocks into larger aggregates by examining the role that entropy plays in this process. Entropic effects are first examined within the confines of a simple model system, and the results are then compared to experimental data on clusters of amino acids. The model system predicts that the formation of a specific structure is more likely to occur from an enantiopure solution because forming a particular structure from a racemic solution is hindered by significant entropic barriers. These predictions are in good agreement with the experimental results. In our examination of clusters of all of the amino acids, clusters which are unusually abundant are found only when enantiopure solutions are sampled. Furthermore, the majority of all clusters exhibit no preference for chiral composition, suggesting that entropic effects negate any changes in enthalpy. Although the experimental data are not comprehensive, our results strongly suggest that specificity in homochiral clusters is entropically advantageous compared to specificity in racemic clusters.

Ion mobility and mass spectrometry techniques have been used to study clusters of serine formed by sonic spray ionization. Broad distributions of cluster sizes and charge states are observed, ranging from clusters containing only a few serine units to clusters that contain more than 600 serine units (i.e., protonated clusters of the form [mSer + nH] n + with m = 8 to >600 and n = 1 to 10). Experimental collision cross sections, derived from the mobility data, are dependent upon cluster size but are not significantly influenced by the cluster charge state. A comparison of calculated cross sections for different trial geometries for several cluster sizes indicates that large clusters adopt tightly packed, roughly spherical geometries. The most abundant cluster size in the spectrum corresponds to the [8Ser + H] + cluster; however, this ion comprises less than 1% of the total serine abundance. The measured cross section, Ω([8Ser + H] + ) = 190 A 2 , is in good agreement with the 191.2 ′ 0.2 A 2 value reported previously from electrospray ionization. Isotopic labeling studies indicate that [8Ser + H] + retains a strong chiroselective preference. Evidence for some chiroselectivity in larger clusters is presented.

Ion mobility/time-of-flight mass spectrometry techniques have been used to examine distributions of fragment ions generated by collision-induced dissociation (CID) in a quadrupole ion trap. The mobility-based separation step prior to mass-to-charge (m/z) analysis reduces spectral congestion and provides information that complements m/z-based assignments of peaks. The approach is demonstrated by examining fragmentation patterns of insulin chain B (a 30-residue peptide), and ubiquitin (a protein containing 76 amino acids). Some fragments of ubiquitin show evidence for multiple stable conformations.

In the structure of DL-proline, C5H9NO2, the molecules are connected via classical intermolecular N-H...O hydrogen bonds involving the amine and carboxyl groups [N...O = 2.7129 (15) and 2.8392 (16) A], and form chains along the b-axis direction and parallel to (-101). The chains are linked into sheets via weak non-classical hydrogen bonds. The conformation of the molecule and its packing are notably different from the monohydrated DL-proline form.

Ion mobility/mass spectrometry techniques are used to study the chiral preferences of small proline clusters (containing 2 to 23 proline monomers) produced by electrospray ionization. By varying the composition of the electrospray solution from enantiomerically pure (100% L or 100% D) to racemic (50:50 L:D), it is possible to delineate which cluster sizes prefer homochiral (resolved) or heterochiral (antiresolved) compositions. The results show a remarkable oscillation in chiral preference. Singly protonated clusters, [xPro+H](+) (where x corresponds to the number of prolines), favor homochiral assemblies (for x = 4, 6, 11 and 12); heterochiral structures are preferred (although the preferences are not as strong) for x = 5 and 7. Larger, doubly protonated clusters [xPro+2H](2+) favor homochiral assemblies for x = 18, 19, and 23 and heterochiral structures for x = 14, 16, 17, 20, 21, and 22. Some of the variations that are observed can be rationalized through simple structures that would lead to especially stable geometries. It is suggested that some antiresolved clusters, such as [22Pro+2H](2+), may be comprised of resolved D- and L-proline domains.

Passive transfer of broadly neutralizing HIV antibodies can prevent infection, which suggests that vaccines that elicit such antibodies would be protective. Thus far, however, few broadly neutralizing HIV antibodies that occur naturally have been characterized. To determine whether these antibodies are part of a larger group of related molecules, we cloned 576 new HIV antibodies from four unrelated individuals. All four individuals produced expanded clones of potent broadly neutralizing CD4-binding-site antibodies that mimic binding to CD4. Despite extensive hypermutation, the new antibodies shared a consensus sequence of 68 immunoglobulin H (IgH) chain amino acids and arise independently from two related IgH genes. Comparison of the crystal structure of one of the antibodies to the broadly neutralizing antibody VRC01 revealed conservation of the contacts to the HIV spike.

A high-capacity ion trap coupled to a time-of-flight (TOF) mass spectrometer has been developed to carry out comprehensive linked scan analysis of all stored ions in the ion trap. The approach involves a novel tapered geometry high-capacity ion trap that can store more than 10 6 ions (range 800–4000 m/z) without degrading its performance. Ions are stored and scanned out from the high-capacity ion trap as a function of m/z, collisionally fragmented and analyzed by TOF. Accurate mass analysis is achieved on both the precursor and fragment ions of all species ejected from the ion trap. We demonstrate the approach for comprehensive linked-scan identification of phosphopeptides in mixtures with their corresponding unphosphorylated peptides. © 2010 Elsevier B.V. All rights reserved.

Chirality plays an important role in the formation and stability of noncovalent clusters which are made of chiral molecules. It is shown that clusters can exhibit a preference for both homochirality and heterochirality. Serine cluster formation is dominated by the formation of heterochiral aggregates, with the exception of the previously observed homochiral serine octamer. Thus, the majority of serine clusters lead to chiral anti-resolution, or the racemic mixing of enantiomers.

Chiral enrichment of serine is achieved in experiments that involve formation of serine octamers starting from non-racemic serine solutions. Serine octamers were generated by means of electrospray and sonic spray ionization of aqueous solutions of d(3)-L-serine (108 Da) and D-serine (105 Da) having different molar ratios of enantiomers. A cyclic process involving the formation of chirally-enriched octameric cluster ions and their dissociation, viz. Ser(1) --Ser(8) --Ser(1), allows serine monomers to be regenerated with increased enantiomeric excess as shown in two types of experiments: (1) Chiral enrichment in serine was observed in MS/MS/MS experiments in a quadrupole ion trap in which the entire distribution of serine octamers formed from non-racemic solutions was isolated, collisionally activated, and fragmented. Monomeric serine was regenerated with increased enantiomeric excess upon dissociation of octamers when compared with the enantiomeric composition of the original solution. (2) Chiral enrichment was observed in the products of soft-landing of mass-selected protonated serine octamers. These ions were generated by means of electrospray or sonic spray ionization, mass selected, and collected on a gold surface using ion soft-landing. Chiral enrichment of the soft-landed serine was established by redissolving the recovered material and comparing the intensities of protonated molecular ions of d(3)-L-serine and D-serine after APCI-MS analysis. Both of these experiments showed comparable results, suggesting that formation of serine octamers depends only on the enantiomeric composition of the serine solution and that the magnitude of the chiral preference is intrinsic to octamers formed from solutions of given chiral composition.

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.

A prototype linear octopole ion trap/ion mobility/tandem mass spectrometer has been coupled with a nanoflow liquid chromatography separation approach and used to separate and characterize a complicated peptide mixture from digestion of soluble proteins extracted from human urine. In this approach, two dimensions of separation (nanoflow liquid chromatography and ion mobility) are followed by collision induced dissociation (CID) and mass spectrometry (MS) analysis. From a preliminary analysis of the most intense CID-MS features in a part of the dataset, it is possible to assign 27 peptide ions which correspond to 13 proteins. The data contain many additional CID-MS features for less intense ions. A limited discussion of these features and their potential utility in identifying complicated peptide mixtures required for proteomics study is presented.

Recent improvements in ion mobility/time-of-flight mass spectrometry techniques have made it possible to incorporate nano-flow liquid chromatography and collision induced dissociation techniques. This combination of approaches provides a new strategy for detailed characterization of complex systems—such as, combinatorial libraries. Our work uses this technology to provide a detailed analysis of a tetrapeptide library having the general form Xxx1-Xxx2-Xxx3-Xxx4 where Xxx1 = Glu, Phe, Val, Asn; Xxx2 = Glu, Phe, Val, Tyr; Xxx3 = Glu, Phe, Val, Thr; and Xxx4 = Glu, Phe, Val, Leu—a system that is expected to contain 256 different peptide sequences. The results corroborate the presence of many expected peptide sequences and indicate that some synthetic steps appear to have failed. Particularly interesting is the observation of a t-butyl protecting group on the tyrosine (Tyr) residue. It appears that most Tyr containing peptides that have this t-butyl group attached favor formation of [2M + 2H]2+ dimers, which can be readily distinguished from [M + H]+ monomers based on differences in their gas-phase mobilities. In this case, we demonstrate the use of the mobility differences between [2M + 2H]2+ and [M + H]+ ions as a signature for a failure of a synthetic step.

Here we show experimental evidence for the spontaneous chiral resolution of icosahedral [12Pro+H]+ cluster ion. Molecular simulations reveal that the icosahedron consists of 12 equally spaced prolines where the rigid pyrrolidine ring of each monomer is sticking out of the closed cage. The tightly packed chiral cage traps a single proton in the center cavity. On the other hand, racemic [12Pro+H]+ cluster size exhibits a prismatic structure that can easily incorporate and lose proline monomeric unit sequentially, thus easily forming other geometries. Mechanisms which account for these observations are discussed.

Mass spectrometry is a method of choice for quantifying low-abundance proteins and peptides in many biological studies. Here, we describe a range of computational aspects of protein and peptide quantitation, including methods for finding and integrating mass spectrometric peptide peaks, and detecting interference to obtain a robust measure of the amount of proteins present in samples.