Peptide-Mass Profiles of Polyvinylidene Difluoride-Bound Proteins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in the Presence of Nonionic Detergents

Gharahdaghi, Farzin; Kirchner, Michele; Fernandez, Joseph; Mische, Sheenah M.

doi:10.1006/abio.1996.0012

Cited by 107 publications

(73 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Control samples consisted of noncross-linked UV-irradiated gp96. The lyophilized samples were dissolved in a matrix solution, which consisted of a saturating amount of ␣-cyano-4-hydroxycinnamic acid in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid (48). The samples (1-2 l) were then spotted on a sample holder plate and allowed to air dry.…”

Section: Matrix-assisted Laser Desorption Time Of Flight Mass Spectromentioning

confidence: 99%

Identification of the Peptide-binding Site in the Heat Shock Chaperone/Tumor Rejection Antigen gp96 (Grp94)

Linderoth¹,

Popowicz²,

Sastry³

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624 -630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by ␣-helices and lies on a surface consisting of antiparallel ␤-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.Specific protective immunity results when heat shock protein (HSP) 1 -peptide complexes purified from tumor cells are injected into inbred mice bearing the same specific type of tumor (1-6). The HSP-mediated specific immunogenicity is also seen with virus-infected cells (7-9). This paradigm is the basis for a new therapeutic strategy against human cancers (9, 10). The immune response is directed against peptides noncovalently bound to the HSP and not to the HSP (for reviews, see Refs. 11 and 12)). HSPs that form the immunogenic peptide complexes include gp96 (GRP94) (13) and calreticulin (14), which reside in the endoplasmic reticulum (ER). Cytosolic HSP70-(15, 16) and HSP90-(17) peptide complexes are also immunogenic. The ER-resident chaperone gp96 (GRP94) has been the most extensively studied from an immunological standpoint (13, 18 -21). It is an abundant stress protein that displays dual functionality: it directs peptides into the immune response pathway and it assists in protein folding. The role of gp96 in the immune response is not well understood at the molecular level. gp96 binds a variety of peptides in vitro and in vivo with little or no apparent amino acid sequence specificity (13,(22)(23)(24). gp96 also binds ATP but the role of nucleotide in peptide loading/unloading is unclear (22,25). Highly purified HSP90, the cytosolic paralog of gp96, probably binds and uses ATP (26). This is in contrast to chaperones HSP70 and BiP (ER paralog of HSP70), where it is clear that ATP binding is important for the release of peptide substrates (r...

show abstract

Section: Matrix-assisted Laser Desorption Time Of Flight Mass Spectromentioning

confidence: 99%

Identification of the Peptide-binding Site in the Heat Shock Chaperone/Tumor Rejection Antigen gp96 (Grp94)

Linderoth¹,

Popowicz²,

Sastry³

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Arginine-containing peptides have been reported to dominate the peptide pattern for protein digests (13 ), the extent of which depends on the matrix used. The presence of ionic detergents such as Triton X-100 and Tween 20 has also been shown to cause signal suppression in MALDI experiments, which can be countered by modifications to the matrix (14 ). Because SELDI also uses the same matrices and ionization process, this technique would be similarly affected.…”

mentioning

confidence: 99%

Ion Suppression in Mass Spectrometry

2003

View full text Add to dashboard Cite

Background: Mass spectrometry (MS) is being introduced into a large number of clinical laboratories. It provides specificity because of its ability to monitor selected mass ions, sensitivity because of the enhanced signal-to-noise ratio, and speed because it can help avoid the need for intensive sample cleanup and long analysis times. However, MS is not without problems related to interference, especially through ion suppression effects. Ion suppression results from the presence of less volatile compounds that can change the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector. Content: This review discusses materials shown to cause ion suppression, including salts, ion-pairing agents, endogenous compounds, drugs, metabolites, and proteins. Experimental protocols for examining ion suppression, which should include, at a minimum, signal recovery studies using specimen extracts with added analyte, are also discussed, and a more comprehensive approach is presented that uses postcolumn infusion of the analyte to evaluate protracted ionization effects. Finally, this review presents options for minimizing or correcting ion suppression, which include enhanced specimen cleanup, chromatographic changes, reagent modifications, and effective internal standardization. Summary: Whenever mass spectrometric assays are developed, ion suppression studies should be performed using expected physiologic concentrations of the analyte under investigation.

show abstract

“…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].…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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.

View full text Add to dashboard Cite

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 ...

show abstract

Peptide-Mass Profiles of Polyvinylidene Difluoride-Bound Proteins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in the Presence of Nonionic Detergents

Cited by 107 publications

References 6 publications

Identification of the Peptide-binding Site in the Heat Shock Chaperone/Tumor Rejection Antigen gp96 (Grp94)

Identification of the Peptide-binding Site in the Heat Shock Chaperone/Tumor Rejection Antigen gp96 (Grp94)

Ion Suppression in Mass Spectrometry

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

Contact Info

Product

Resources

About