Colin S. Creaser

Ion mobility spectrometry: a review. Part 1. Structural analysis by mobility measurement

Ion mobility spectrometry (IMS) is an electrophoretic technique that allows ionised analyte molecules to be separated on the basis of their mobilities in the gas phase. The technique has found widespread application as a detector, most noticeably for chemical warfare agents on the battlefield and for explosives and narcotics at ports and airports. The application of IMS to structural studies of small molecules has also been recognised since the advent of the technique in the 1970s. The coupling of IMS with electrospray (ESI) and MALDI ion sources has opened up exciting possibilities for the study of the conformations and structures of a wide range of biomolecules in the gas-phase, including proteins, peptides and oligonucletoides. This tutorial review discusses the principles, instrumentation and applications of IMS for the elucidation of molecular structural characteristics.

Serum Proteomic Fingerprinting Discriminates Between Clinical Stages and Predicts Disease Progression in Melanoma Patients

PURPOSE Currently known serum biomarkers do not predict clinical outcome in melanoma. S100-beta is widely established as a reliable prognostic indicator in patients with advanced metastatic disease but is of limited predictive value in tumor-free patients. This study was aimed to determine whether molecular profiling of the serum proteome could discriminate between early- and late-stage melanoma and predict disease progression. PATIENTS AND METHODS Two hundred five serum samples from 101 early-stage (American Joint Committee on Cancer [AJCC] stage I) and 104 advanced stage (AJCC stage IV) melanoma patients were analyzed by matrix-assisted laser desorption/ionisation (MALDI) time-of-flight (ToF; MALDI-ToF) mass spectrometry utilizing protein chip technology and artificial neural networks (ANN). Serum samples from 55 additional patients after complete dissection of regional lymph node metastases (AJCC stage III), with 28 of 55 patients relapsing within the first year of follow-up, were analyzed in an attempt to predict disease recurrence. Serum S100-beta was measured using a sandwich immunoluminometric assay. RESULTS Analysis of 205 stage I/IV serum samples, utilizing a training set of 94 of 205 and a test set of 15 of 205 samples for 32 different ANN models, revealed correct stage assignment in 84 (88%) of 96 of a blind set of 96 of 205 serum samples. Forty-four (80%) of 55 stage III serum samples could be correctly assigned as progressors or nonprogressors using random sample cross-validation statistical methodologies. Twenty-three (82%) of 28 stage III progressors were correctly identified by MALDI-ToF combined with ANN, whereas only six (21%) of 28 could be detected by S100-beta. CONCLUSION Validation of these findings may enable proteomic profiling to become a valuable tool for identifying high-risk melanoma patients eligible for adjuvant therapeutic interventions.

Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis.

A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.

High‐throughput ultra‐high‐performance liquid chromatography/tandem mass spectrometry quantitation of insulin‐like growth factor‐I and leucine‐rich α‐2‐glycoprotein in serum as biomarkers of recombinant human growth hormone administration

Insulin-like growth factor-I (IGF-I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine-rich alpha-2-glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5-min ultra-high-performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)-based selected reaction monitoring (SRM) assay detected both IGF-I and LRG at endogenous concentrations. Four eight-point standard addition curves of IGF-I (16-2000 ng/mL) demonstrated good linearity (r(2) = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS-derived IGF-I concentrations correlated well against immunochemistry-derived values. Combining IGF-I and LRG data improved the separation of treated and placebo states compared with IGF-I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF-I data demonstrated an improved model over that developed using IGF-I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS-based quantitation of a medium abundance protein (IGF-I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF-I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS.

The use of shift reagents in ion mobility-mass spectrometry: studies on the complexation of an active pharmaceutical ingredient with polyethylene glycol excipients

Gas-phase ion mobility studies of mixtures containing polyethylene glycols (PEG) and an active pharmaceutical ingredient (API), lamivudine, have been carried out using electrospray ionization-ion mobility spectrometry-quadrupole-time-of-flight mass spectrometry (ESI-IMS-Q-TOF). In addition to protonated and cationized PEG oligomers, a series of high molecular weight ions were observed and identified as noncovalent complexes formed between lamivudine and PEG oligomers. The noncovalent complex ions were dissociated using collision induced dissociation (CID) after separation in the ion mobility drift tube to recover the protonated lamivudine free from interfering matrix ions and with a drift time associated with the precursor complex. The potential of PEG excipients to act as “shift reagents,” which enhance selectivity by moving the mass/mobility locus to an area of the spectrum away from interferences, is demonstrated for the analysis of lamivudine in a Combivir formulation containing PEG and lamivudine.

Acceptance criteria for analytical data on polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans

Abstract Guidelines for the validation and quality control of analytical work related to polychlorinated dibenzo-p-dioxins and dibenzofurans are presented. The intention is that these guidelines should be used to help to ensure the reliability and comparability of results, especially those which may be used as a basis for advising Government.

Immunoaffinity chromatography combined on-line with high-performance liquid chromatography–mass spectrometry for the determination of corticosteroids

On-line coupled immunoaffinity chromatography-reversed-phase high-performance liquid chromatography (IAC-HPLC) with detection by quadrupole ion trap mass spectrometry using a particle beam interface has been developed for the determination of the steroids, dexamethasone and flumethasone. HEMA (polyhydroxyethylmethacrylate) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. Antibody cross-reactivity and non-specific binding have been investigated for the HEMA bound anti-dexamethasone IAC column. The on-line IAC-HPLC-MS determination of dexamethasone and flumethasone in post-administration equine urine samples showed precisions (R.S.D.) of 8.0 and 7.1%, respectively, with limits of detection in the range 3-4 ng/ml.

Survey of background levels of PCDDs & PCDFs in UK soils

Abstract Soil samples collected at points on a 50 km grid covering England, Wales and lowland Scotland have been analysed for PCDDs and PCDFs. Mean concentrations for a reduced data set were in the range 6.6 ng kg −1 for PCDD to 191 ng kg −1 for OCDD and from 23 ng kg −1 for PCDF to 41 ng kg −1 for HxCDF. These levels provide an indication of the background concentrations of PCDDs and PCDFs in British soils.

Levels and sources of PCDDs and PCDFs in urban British soils.

Soil samples from five British cities have been analysed for PCDDs and PCDFs. Mean concentrations for PCDD and PCDF congeners were: TCDD, 65 ng Kg−2; PeCDD, 69 ng Kg−1; HxCDD, 154 ng Kg−1; HpCDD, 817 ng Kg−1; OCDD, 9980 ng Kg−1; TCDF, 232 ng Kg−1; PeCDF, 189 ng Kg−1; HpCDF, 156 ng Kg−1; HpCDF, 152 ng Kg−1; and OCDF, 196 ng Kg−1. The mean levels for urban samples are significantly higher (p < 0.001) than rural and semi-urban samples previously reported. Principal component analysis of isomer distributions for urban samples and those of known origin indicate that combustion processes are the principal source of PCDDs and PCDFs in these soils.

Diagnostic biomarkers differentiating metastatic melanoma patients from healthy controls identified by an integrated MALDI-TOF mass spectrometry/bioinformatic approach

The prognosis of advanced metastatic melanoma (American Joint Committee on Cancer (AJCC) stage IV) remains dismal with a 5‐year survival rate of 6–18%. In the present study, an integrated MALDI mass spectrometric approach combined with artificial neural networks (ANNs) analysis and modeling has been used for the identification of biomarker ions in serum from stage IV melanoma patients allowing the discrimination of metastatic disease from healthy status with high specificities of 92% for protein ions and 100% for peptide biomarkers. Our ANNs model also correctly classified 98% of a blind validation set of AJCC stage I melanoma samples as nonstage IV samples, emphasizing the power of the newly defined biomarkers to identify patients with late‐stage metastatic melanoma. Sequence analysis identified peptides derived from metastasis‐associated proteins; alpha 1‐acid glycoprotein precursor‐1/2 (AAG‐1/2) and complement C3 component precursor‐1 (CCCP‐1). Furthermore, quantitation of serum AAG by an immunoassay showed a significant (p<0.001) increase in AAG serum concentration in stage IV patients in comparison with healthy volunteers; moreover; the quantity of AAG plotted against MALDI‐MS peak intensity classified the groups into two distinct clusters. Ongoing studies of other disease stages will provide evidence whether our strategy is sufficiently robust to give rise to stage‐specific protein/peptide signatures in melanoma.