We report the coupling of a hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), to a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS). ...The details of the source design and initial data are presented. Analysis of peptides and proteins ranging from 1 to 8.6 kDa resulted in high resolving power single-acquisition FT-ICR mass spectra with average charge-states highly correlated to those obtained by nanoESI, thus, providing strong evidence that the ESI process dictates the observed charge-state distribution. Importantly, unlike the recently introduced electrospray assisted laser desorption ionization (ELDI) source reported by Shiea and coworkers
1, 2, the data we have obtained to date rely on the use of an organic acid matrix. The results presented herein provide insight into the charging mechanism of this emerging ionization approach, while also expanding the utility of FT-ICR MS for top-down protein and complex mixture analysis.
We report the implementation of an infrared laser onto our previously reported matrix-assisted laser desorption electrospray ionization (MALDESI) source with ESI post-ionization yielding multiply ...charged peptides and proteins. Infrared (IR)-MALDESI is demonstrated for atmospheric pressure desorption and ionization of biological molecules ranging in molecular weight from 1.2 to 17 kDa. High resolving power, high mass accuracy single-acquisition Fourier transform ion cyclotron resonance (FT-ICR) mass spectra were generated from liquid- and solid-state peptide and protein samples by desorption with an infrared laser (2.94 μm) followed by ESI post-ionization. Intact and top-down analysis of equine myoglobin (17 kDa) desorbed from the solid state with ESI post-ionization demonstrates the sequencing capabilities using IR-MALDESI coupled to FT-ICR mass spectrometry. Carbohydrates and lipids were detected through direct analysis of milk and egg yolk using both UV- and IR-MALDESI with minimal sample preparation. Three of the four classes of biological macromolecules (proteins, carbohydrates, and lipids) have been ionized and detected using MALDESI with minimal sample preparation. Sequencing of O-linked glycans, cleaved from mucin using reductive β-elimination chemistry, is also demonstrated.
We present solid- and liquid-state IR-MALDESI coupled to FT-ICR mass spectrometry for intact and top-down characterization of biomolecules and direct analysis.
The presence of estrogen receptor (ER) in breast cancer is a prognostic indicator for both disease-free and overall survival. 16α-(18)F-fluoro-17β-estradiol ((18)F-FES) with PET is a noninvasive test ...for evaluation of ER expression and has been used for predicting response to endocrine therapy in patients with ER-positive metastatic breast cancer. The purpose of this study was to correlate (18)F-FES PET and ER expression in patients with primary, operable breast cancer.
Forty-eight patients were prospectively enrolled in an institutional review board-approved protocol and signed an informed consent form. All patients had undergone (18)F-FES PET preoperatively. Clinical characteristics, tumor characteristics, and treatment outcomes were recorded. Immunohistochemical analysis for ER and progesterone receptor (PgR) percentage expression (46 surgical, 2 core biopsy specimens) was performed. (18)F-FES PET standardized uptake value (SUV) of the breast lesion was correlated with percentage immunohistochemistry ER and PgR expression. (18)F-FES PET SUV was quantified, with a value of 1.5 or more considered positive, and ER and PgR was quantified, with 1% or more considered positive. Formalin-fixed paraffin-embedded tissue was available for 44 patients (42 surgical, 2 core biopsy specimens). We used a microarray platform, and estrogen-related gene expression data (ESR1, ESR2, and PGR) were compared with (18)F-FES PET SUV (Spearman rank correlation). Tumor size, ductal histology, grade, HER2-neu overexpression, PgR expression, estradiol level, body mass index (BMI), and lean BMI were compared with (18)F-FES PET uptake using univariate and multivariate analysis.
Forty-eight patients completed our protocol, and 2 patients did not undergo surgery because bone metastases were identified preoperatively on (18)F-FES PET. Eighty-three percent of our patients were stage I or II, with a median tumor size of 1.9 cm. Forty-one patients underwent a sentinel node biopsy. Twenty-one patients had nodal involvement. (18)F-FES PET identified 5 patients with axillary nodal uptake (median SUV, 3.0; range, 1.7-6.9). These 5 patients had ER-positive breast cancer, and all had more than 4 positive nodes at the time of axillary node dissection. (18)F-FES PET SUV was associated with immunohistochemistry ER expression. The sensitivity and specificity of the (18)F-FES PET for the breast lesion were 0.85 and 0.75, respectively. Estrogen and progesterone gene expression (ESR1, ESR2, and PGR) was not associated with (18)F-FES PET SUV (Spearman rank correlation). We found a significant correlation between (18)F-FES PET SUV and tumor size (P = 0.0015) but not with ductal histology, grade, HER2-neu overexpression, PgR, estradiol, BMI, or lean BMI (logistic regression). ER expression (P < 0.001) and tumor size (P < 0.0001) were significant on multivariate regression analysis.
(18)F-FES PET SUV correlated with ER immunohistochemistry expression but not gene expression in our patients with early breast cancer. We found that size of primary tumor was significantly associated with (18)F-FES PET SUV. (18)F-FES PET is highly predictive for metastatic disease and helped in the identification of patients with metastatic disease in a preoperative setting.
We have developed an atmospheric pressure ionization technique called liquid matrix-assisted laser desorption electrospray ionization (liq-MALDESI) for the generation of multiply charged ions by ...laser desorption from liquid samples deposited onto a stainless steel sample target biased at a high potential. This variant of our previously reported MALDESI source does not utilize an ESI emitter to postionize neutrals. Conversely, we report desorption and ionization from a macroscopic charged droplet. We demonstrate high mass resolving power single-acquisition FT-ICR-MS analysis of peptides and proteins ranging from 1 to 8.6 kDa at atmospheric pressure. The liquid sample acts as a macroscopic charged droplet similar to those generated by electrospray ionization, whereby laser irradiation desorbs analyte from organic matrix containing charged droplets generating multiply charged ions. We have observed a singly charged radical cation of an electrochemically active species indicating oxidation occurs for analytes and therefore water; the latter would play a key role in the mechanism of ionization. Moreover, we demonstrate an increase in ion abundance and a concurrent decrease in surface tension with an increase in the applied potential.
The use of aerodynamic devices in ambient ionization source development has become increasingly prevalent in the field of mass spectrometry. In this study, an air ejector has been constructed from ...inexpensive, commercially available components to incorporate an electrospray ionization emitter within the exhaust jet of the device. This novel aerodynamic device, herein termed remote analyte sampling, transport, and ionization relay (RASTIR) was used to remotely sample neutral species in the ambient and entrain them into an electrospray plume where they were subsequently ionized and detected using a linear ion trap Fourier transform mass spectrometer. Two sets of experiments were performed in the ambient environment to demonstrate the deviceʼs utility. The first involved the remote (∼1 ft) vacuum collection of pure sample particulates (i.e., dry powder) from a glass slide, entrainment and ionization at the ESI emitter, and mass spectrometric detection. The second experiment involved the capture (vacuum collection) of matrix-assisted laser desorbed proteins followed by entrainment in the ESI emitter plume, multiple charging, and mass spectrometric detection. This approach is in principle a RASTIR-assisted matrix-assisted laser desorption electrospray ionization source (Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712−1716; Rapid Commun. Mass Spectrom. 2007, 21, 1150−1154.). A detailed description of the device construction, operational parameters, and preliminary small molecule and protein data are presented.
The design and implementation of a radio frequency acoustic desorption ionization (RADIO) source has been demonstrated for the analysis of multiply charged peptides and proteins. One μL aliquots of ...melittin, BNP-32, and ubiquitin (∼1 μg of analyte) were deposited onto a quartz crystal microbalance (QCM) electrode before radio frequency actuation for desorption. Continuous electrospray parallel to/above the sampling surface enabled the ionization of desorbed species. Detection by a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer confirmed the intact and dissociated species observed during MS and MS/MS experiments, respectively.
The development of a
radio frequency
acoustic
desorption
ionization (RADIO) source has resulted in the MS and MS/MS analysis of multiply-charged peptides and proteins.
The design and construction of a high precision ambient ionization source matrix-assisted laser desorption electrospray ionization (MALDESI) are described in full detail, including a complete parts ...list. The computer controlled high precision motion control system and high repetition rate Explorer laser are demonstrated during MALDESI-FT-ICR analysis of peptides and proteins ranging from 1 to 17 kDa. The high stability ionization source platform described herein demonstrates both the advantages of the new MALDESI source and versatility for application to numerous desorption and ionization techniques.
We present the development of a high precision ionization platform for direct analysis and high spatial resolution ambient imaging mass spectrometry.
PDF
