Efficacy and compatibility with mass spectrometry of methods for elution of proteins from sodium dodecyl sulfate–polyacrylamide gels and polyvinyldifluoride membranes

Jørgensen, Charlotte Sværke; Jagd, Mitchell; Sørensen, Birgitte Kjær; McGuire, Jim; Barkholt, Vibeke; Højrup, Peter

doi:10.1016/j.ab.2004.03.012

Cited by 28 publications

(17 citation statements)

References 34 publications

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“…However, in SDS-PAGE estimation of relative molecular mass (M r ) is based on comparison with reference proteins and accuracy of determined M r is relatively poor with 65-10% [10][11][12]. When proteins are electroeluted or electroblotted from slab gels and submitted either to electrospray ionizationmass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization (MALDI)-MS subsequently [12][13][14], mass accuracy improves to ,0.01-0.6% [15,16], but the methodology still suffers from critical selection of mass calibrants [15], interferences with buffer compounds, detergents [13,15], and staining compounds [10,15]. Sensitivity levels off at gel-loadings , 25 pmol due to extraction losses [15], which are probably related to the staining conditions that denature proteins [5].…”

Section: Introductionmentioning

confidence: 99%

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

2005

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High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.

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Section: Introductionmentioning

confidence: 99%

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

2005

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“…This growth has resulted in mass spectrometry being used in tandem with a variety of other experimental techniques [3][4][5]. However, many of these techniques utilize reagents that can adversely affect the quality of mass spectra obtained by either suppressing analyte ionization or causing the formation of numerous adducts [6]. To avoid these effects, it is usually necessary to pretreat biological samples before analysis [7], a process which can lead to significant loss of analyte.…”

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The effect of Tween80 on signal intensity of intact proteins by MALDI time-of-flight mass spectrometry

Brinkworth

Bourne

2007

J. Am. Soc. Mass Spectrom.

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Buffers and detergents are notorious for suppression of analyte signal in electrospray and MALDI mass spectrometry and, invariably, analysts will take steps to remove these contaminants before MS analysis. However, we have found serendipitously that protein signal with MALDI MS is improved by about an order of magnitude on the addition of small amounts of Tween80. Four charged states of BSA could easily be seen at less than 125 fmol/spot and with mixture of three proteins (BSA, trypsinogen, and protein A) the molecular ions could be detected on as little as 12.5 fmol of spotted material (per protein) using an automated laser firing sequence. T he versatility of mass spectrometry for the identification and characterization of proteins continues to grow [1,2]. This growth has resulted in mass spectrometry being used in tandem with a variety of other experimental techniques [3][4][5]. However, many of these techniques utilize reagents that can adversely affect the quality of mass spectra obtained by either suppressing analyte ionization or causing the formation of numerous adducts [6]. To avoid these effects, it is usually necessary to pretreat biological samples before analysis [7], a process which can lead to significant loss of analyte. In addition, it has been reported that modification of the matrix solution can be one way to suppress these effects such that MALDI-TOF MS can be obtained in the presence of nonionic detergents [8].Our interest was to develop a confirmatory test for the presence of low level amounts of proteins using mass spectrometry. In addition, the method had to accommodate samples containing Tween80, a nonanionic detergent used in the preparation of biological buffers that assists in the solubilization of proteins [9]. Amini and coworkers [10] reported that Tween80 produced prominent spectra of its own in the presence of sodium or potassium ions to the detriment of analyte signal.To develop our methodology for low level protein detection, we used a model protein, bovine serum albumin (BSA), and were surprised to find that the addition of Tween80, rather than leading to signal degradation, produced the opposite result. In this communication, we discuss the unexpected advantageous effect of Tween80 on the MALDI spectrum of large proteins. Materials and Methods␣-Cyano-4-hydroxycinammic acid (cat. no. 70990) and sinapic acid (cat. no. 85429) were obtained from Fluka (St. Louis, MO) and were both in excess of 99.6% pure. HPLC grade water was from Merck (Darmstadt, Germany). Tween80 was from Sigma (St. Louis, MO). Bovine serum albumin, high purity, was from Pharmacia (New York, NY). Protein calibration standard II (trypsinogen, protein A, and bovine serum albumin mix) was from Bruker Daltonik (Leipzig, Germany). Preparation of Solutions of Bovine Serum Albumin (BSA) and Protein Calibration Standard IIAll the relevant stock and working solutions for each set of comparison experiments were prepared, spotted, and the mass spectra acquired on the same day to ensure, as much as possible, that the only ...

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“…Molecular markers that can potentially be used to identify small lesions that are invisible to imaging techniques could provide an opportunity to treat a neoplasm before it invades tissue. Markers that can detect ductal carcinoma in situ (DCIS) 6 would be particularly valuable because nearly 100% of women diagnosed at this early stage of breast cancer can be cured.…”

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Independent Validation of Candidate Breast Cancer Serum Biomarkers Identified by Mass Spectrometry

Li¹,

Orlandi²,

White³

et al. 2005

Clinical Chemistry

161

159

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Background:We previously selected a panel of 3 breast cancer biomarkers (BC1, BC2, and BC3) from serum samples collected at a single hospital based on their collective contribution to the optimal separation of breast cancer patients and noncancer controls by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The identities and general applicability of these markers, however, were unknown. In this study, we performed protein expression profiling on samples obtained from a second hospital, included a greater number of ductal carcinoma in situ (DCIS) cases, and performed purification and identification of the 2 confirmed markers. Methods: Using a case-control study design, we performed protein expression profiling on serum samples from the National Cancer Institute (Milan, Italy). The validation sample cohort consisted of 61 women with locally invasive breast cancer, 32 with DCIS, 37 with various benign breast diseases (including 13 atypical), and 46 age-matched apparently healthy women (age range, 44 -68 years). Validated biomarkers were purified and identified with serial chromatography, 1-dimensional gel electrophoresis, in-gel ASP-N digestion, peptide mass fingerprinting, and tandem mass peptide sequencing. Results: The BC3 and BC2 expression patterns in this sample set were consistent with the first study sample

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Efficacy and compatibility with mass spectrometry of methods for elution of proteins from sodium dodecyl sulfate–polyacrylamide gels and polyvinyldifluoride membranes

Cited by 28 publications

References 34 publications

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

The effect of Tween80 on signal intensity of intact proteins by MALDI time-of-flight mass spectrometry

Independent Validation of Candidate Breast Cancer Serum Biomarkers Identified by Mass Spectrometry

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