Biomedical subjects

Amy E Hilderbrand

Publications and source records attributed to Amy E Hilderbrand.

8 recordsLinked to original sources

PubMed2006

Exploring crown ethers as shift reagents for ion mobility spectrometry.

A series of crown ethers, 12-crown-4, 15-crown-5, 18-crown-6, and dibenzo-30-crown-10, are examined as a possible means of shifting the mobilities of peptide ions. In this approach, a crown ether is added to a solution containing a mixture of peptides and is electrosprayed into the gas phase in order to create distributions of peptide-crown complexes. The ion complexes have different mobilities than the naked peptide ions, and the crown ether molecules appear to interact specifically with basic sites in the peptides thus providing some sequence selectivity. After the peptide-crown complexes are separated by ion mobility spectrometry, the ions can be collisionally activated to dissociate the complex (forming the naked peptide ions) prior to m/z analysis. The overall effect is that complex formation shifts peptide ions to different regions of the mobility spectrum, extending the ability to resolve components. The approach is illustrated by examining isobaric dipeptides as well as a combinatorial library containing 27 tripeptides. Cross sections for the series of crown ether ions and complexes that are observed are reported.

Crown Ethers↗

PubMed2005

Determination of sequence-specific intrinsic size parameters from cross sections for 162 tripeptides.

Ion mobility and mass spectrometry techniques have been used to measure cross sections for 162 tripeptide sequences (27 different sets of six sequence isomers). The isomers have the general forms ABC, ACB, BAC, BCA, CAB, and CBA, where A corresponds to the amino acids Asp, Glu, or Gly, B corresponds to Lys, Arg, or Leu, and C corresponds to Phe, Tyr, or Ser. From these data, we derive a set of size parameters for individual amino acids that reflect the position of the amino acid in the sequence. These sequence-specific intrinsic size parameters (SSISPs) are used to retrodict cross-section values for the 162 measured sequences and to predict cross sections for all remaining tripeptide sequences (567 different sequences) that are comprised of these residues. In several types of peptide compositions, the position of the amino acid in the sequence has a significant impact on the parameter that is derived. For example, the sequence-specific intrinsic size parameter for leucine in the third position of a peptide (SSISP(Leu3)) is approximately 10% larger than SSISP(Leu1). On average, cross sections that are derived using SSISPs provide a better representation of the experimental value than those derived from composition only intrinsic size parameters, derived as described previously (Valentine et al. J. Phys. Chem. 1999, 103, 1203). Finally, molecular modeling techniques are used to derive some insight into the origin of cross-section differences that arise from sequence variation.

Amino Acid Sequence↗

PubMed2005

Developing liquid chromatography ion mobility mass spectometry techniques.

When a packet of ions in a buffer gas is exposed to a weak electric field, the ions will separate according to differences in their mobilities through the gas. This separation forms the basis of the analytical method known as ion mobility spectroscopy and is highly efficient, in that it can be carried out in a very short time frame (micro- to milliseconds). Recently, efforts have been made to couple the approach with liquid-phase separations and mass spectrometry in order to create a high-throughput and high-coverage approach for analyzing complex mixtures. This article reviews recent work to develop this approach for proteomics analyses. The instrumentation is described briefly. Several multidimensional data sets obtained upon analyzing complex mixtures are shown in order to illustrate the approach as well as provide a view of the limitations and required future work.

Animals↗

PubMed2003

Development of high-sensitivity ion trap ion mobility spectrometry time-of-flight techniques: a high-throughput nano-LC-IMS-TOF separation of peptides arising from a Drosophila protein extract.

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.

Animals↗

PubMed2003

Development of LC-IMS-CID-TOFMS techniques: analysis of a 256 component tetrapeptide combinatorial library.

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.

Amino Acid Sequence↗

PubMed2002

Development of high-throughput liquid chromatography injected ion mobility quadrupole time-of-flight techniques for analysis of complex peptide mixtures.

The development of a multidimensional approach involving high-performance liquid chromatography (LC), ion mobility spectrometry (IMS) and tandem mass spectrometry is described for the analysis of complex peptide mixtures. In this approach, peptides are separated based on differences in their LC retention times and mobilities (as ions drift through He) prior to being introduced into a quadrupole/octopole/time-of-flight mass spectrometer. The initial LC separation and IMS dispersion of ions is used to label ions for subsequent fragmentation studies that are carried out for mixtures of ions. The approach is demonstrated by examining a mixture of peptides generated from tryptic digestion of 18 commercially available proteins. Current limitations of this initial study and potential advantages of the experimental approach are discussed.

Amino Acid Sequence↗

PubMed2002

Resolving isomeric peptide mixtures: a combined HPLC/ion mobility-TOFMS analysis of a 4000-component combinatorial library.

A reversed-phase high-performance liquid chromatography (HPLC) separation approach has been combined with ion mobility/time-of-flight (TOF) mass spectrometry in order to characterize a combinatorial peptide library designed to contain 4000 peptides of the general form NH2-Xxx-Xxx-XXX-CO2H, NH2-Ala-Xxx-Xxx-Xxx-CO2H, NH2-Ser-Ala-Xxx-Xxx-Xxx-CO2H and NH2-Leu-Ser-Ala-Xxx-Xxx-Xxx-CO2H (where Xxx represents a randomization over 10 different amino acids: Ala, Arg, Asp, Glu, Gly, Leu, Lys, Phe, Ser, and Val). Addition of the gas-phase mobility separation between the HPLC separation and TOF measurement dimensions makes it possible to resolve many peptide isomers that have identical retention times (and masses).

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Nanoflow LC/ion mobility/CID/TOF for proteomics: analysis of a human urinary 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.

Amino Acid Sequence↗