Capillary Array Electrophoresis-MALDI Mass Spectrometry Using a Vacuum Deposition Interface

Preisler, Jan; Hu, Ping; Rejtar, Tomáš; Moskovets, Eugene; Karger, Barry L.

doi:10.1021/ac010692p

Cited by 89 publications

(94 citation statements)

References 44 publications

(60 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Analysis of the total cell digest has the advantage of minimizing losses of low level proteins, as well as reducing the complexities of the many sample aliquots that can result from approaches such as organelle isolation and gel based studies. In related studies it has been shown that coupling of either ESI [31] or MALDI [32] to a separation system gives a significant increase in the number of peptide or protein identifications. This improved result was attributed to the use of a prefractionation step to minimize the selective ionization of certain solutes that is often observed in direct MS measurements of complex mixtures.…”

Section: Discussionmentioning

confidence: 99%

An approach to the proteomic analysis of a breast cancer cell line (SKBR‐3)

Wu¹,

Hancock²,

Goodrich

et al. 2003

Proteomics

View full text Add to dashboard Cite

An approach to the proteomic analysis of a breast cancer cell line (SKBR-3)This report describes the profiling of proteins in a sample prepared by laser capture microdissection (LCM) from a breast cancer cell line (SKBR-3). This experimental approach serves as a model system for proteomic studies on selected tissue samples and for studies of specific cell types. The captured cells were isolated in a dehydrated and reduced state and solubilized with a denaturing buffer. After dilution the protein mixture was digested with trypsin and the resulting peptide mixture was fractionated by reversed phase HPLC (RPLC) and analyzed on an ion trap mass spectrometer. A key part of this study is the combination of the LCM process with an extraction/digestion procedure that allowed effective solubilization of a significant part of the cellular sample in a single step. The identity of the peptides was determined by tandem mass spectrometry measurements in which the resulting spectra were compared with genomic and proteomic databases and protein identifications were made. While only peptides with a high probability assignment were used, the interpretation of mass spectral fragmentation patterns were also confirmed by manual interpretation of the spectra. Also, for the more abundant proteins the initial protein assignment from the best match peptide was strengthened by the observation of additional confirmatory peptide identifications. Another selection criteria was correlation of the mass spectrometric studies with clinical and genomic studies of potential cancer markers in tumor samples. This proteomic study allowed identification of the following proteins: human receptor protein kinase HER-2 or ERBB-2 and related kinases HER-3 and HER-4, the gene products from breast cancer type I and II susceptibility genes and cytoskeletal components such as cytokeratins 8, 18 and 19. Other proteins include fibroblast growth factor receptor variants (FGFR-2&4) and T-lymphoma invasion and metastasis inducing protein 1 (TIAM1). In addition several nonreceptor protein kinases YES, FAK and JAK-1 and 3 were identified. Since the study was performed on a limited number of cells (approximately 10 000) it raises the possibility of such studies being performed on individual patient samples prepared by needle biopsy.

show abstract

Section: Discussionmentioning

confidence: 99%

An approach to the proteomic analysis of a breast cancer cell line (SKBR‐3)

Wu¹,

Hancock²,

Goodrich

et al. 2003

Proteomics

View full text Add to dashboard Cite

show abstract

“…The peptides of the combined calibration mixtures 1 and 2 were dissolved in 20% ACN/0.01% TFA to final concentrations of 125 fmol/ L des-arg 1 -bradykinin, 413 fmol/ L angiotensin I, 163 fmol/ L glu 1 -fibrinopeptide B, 6 fmol/ L neurotensin, 250 fmol/ L ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), 188 fmol/ L ACTH (18 -39), 375 fmol/ L ACTH , and 438 fmol/ L bovine insulin. The in-solution BSA digest was dissolved in 30% ACN/0.1% TFA to a final concentration of 1 fmol/ L. p-Methoxybenzylpyridinium chloride, which was used for the measurements of the effective temperatures, was dissolved in 20% ACN to final concentrations of 2 and 4 pmol/ L. The initial velocities were measured from 350 fmol bovine insulin as analyte (c ϭ 700 fmol/ L, V ϭ 0.5 L) dissolved in 20% ACN/0.01% TFA; 0.5 L matrix solution (30% ACN/0.1% TFA, c ϭ 45 mM for SA; 70% ACN/0.1% TFA, c ϭ 30 mM for all other matrices) and 0.5 L analyte solution were mixed directly on a polished steel target and air-dried for standard DD preparation.…”

Section: Sample Preparation and Measurementmentioning

confidence: 99%

Comparison between vacuum sublimed matrices and conventional dried droplet preparation in MALDI-TOF mass spectrometry

Jaskolla

Karas

Roth

et al. 2009

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

The properties of several cinnamic acid compounds used as matrices for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were investigated as standard dried droplet (DD) and vacuum sublimed preparations. The differences between both preparation methods were analyzed with regard to matrix grain size, internal ion energy, initial velocity, analyte intensity, and analyte incorporation depth. Some of the used cinnamic acid derivatives exhibit clearly reduced grain sizes as sublimed preparations compared with standard DD approaches. In these cases higher effective temperatures could be measured accompanied by increased analyte intensities, which can be explained by stronger volatilization processes caused by a hindered heat dissipation resulting in a raised analyte transfer into the gas phase. For all sublimed compounds, a strong increase of the initial ion velocity compared with DD preparations could be measured. Higher initial ion velocities correlate with a decrease in internal ion energy which might be attributed to the very uniform crystal morphology exhibited by sublimed compounds. For sublimed matrices without reduced grain size, at least slightly higher analyte intensities could be detected at raised laser fluences. Analyte accumulation in the uppermost matrix layers or the detected higher ion stability can be explanations for these results. Ever increasing numbers of samples call for automation in high-throughput workflows. One of the key areas for progress in automated MALDI analyses consists of improved sample preparation techniques. Numerous efforts have been conducted for improving the preparation step, e.g., the generation of thin uniform matrix layers with high shot-to-shot reproducibility using the aerospray technique by combination of matrix and nitrocellulose [4] or for analysis of synthetic polymers [5]. Also, electrospray deposition and vacuum deposition were reported to yield homogeneous sample preparations with increased analyte intensities [6 -9]. Matrix deposition by sublimation for MALDI imaging has been reported to yield more intense signals with less alkali adducts compared with the electrospray technique [10]. Additionally, sublimed matrix spots deposited on extremely hydrophobic surfaces described in [11][12][13][14] offer a variety of beneficial properties compared with standard dried droplet (DD) preparations: The main advantage of sublimed matrix spots compared with standard DD preparations are higher analyte sensitivities [14 -17]. This is likely related to the formation of considerably smaller crystals and narrow size distribution of thin-film sublimed matrices, such as ␣-cyano-4-hydroxycinnamic acid (CHCA), compared with DD preparations with comparatively large particle sizes and broader size distribution [15]. As a consequence of this, the specific matrix surface area as well as the amount of potential binding places increases compared with standard DD precipitates, which will result in increased analyte binding properties. Additionally, th...

show abstract

“…[67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83] Only these two configurations are covered in this review, although, brief descriptions of the other three are given in the succeeding paragraphs.…”

Section: Ionization Methodsmentioning

confidence: 99%

“…This approach was extended to a capillary array system. 76 Ørsnes et al 67 constructed an interface based on a rotating ball inlet (ROBIN), in which the sample adhering to the surface of the ball is continuously carried past a polymer gasket into the vacuum chamber. Musyimi et al 83 modified this design so that the majority of the ball surface is at atmospheric pressure and easily accessible for sample deposition.…”

Section: ·2 Maldi-time-of-flight(tof)-msmentioning

confidence: 99%

Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides

Monton

Terabe

2005

ANAL. SCI.

View full text Add to dashboard Cite

Many researchers have invested considerable efforts toward improving capillary electrophoresis (CE)-mass spectrometry (MS) systems so they can be applied better to standard analyses. This review highlights the developments in CE-MS of proteins and peptides over the last five years. It includes the developments in interfaces, sample-enrichment techniques, microfabricated devices, and some applications, largely in capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and capillary isotachophoresis formats.

show abstract

Capillary Array Electrophoresis-MALDI Mass Spectrometry Using a Vacuum Deposition Interface

Cited by 89 publications

References 44 publications

An approach to the proteomic analysis of a breast cancer cell line (SKBR‐3)

An approach to the proteomic analysis of a breast cancer cell line (SKBR‐3)

Comparison between vacuum sublimed matrices and conventional dried droplet preparation in MALDI-TOF mass spectrometry

Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides

Contact Info

Product

Resources

About