Two-Layer Sample Preparation:  A Method for MALDI-MS Analysis of Complex Peptide and Protein Mixtures

Dai, Yichen; Whittal, Randy M.; Liang, Li

doi:10.1021/ac980684h

Cited by 201 publications

(151 citation statements)

References 23 publications

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“…For peptide mass mapping by MALDI time-of-flight (TOF) MS, a two-layer sample/matrix preparation method°was°used° [37]°with°␣-cyano-4-hydroxy-cinnamic acid (HCCA) as matrix. 0.7 l of the first-layer matrix solution containing 12 mg/ml of HCCA in 20% methanol/acetone was deposited on the MALDI target and air-dried.…”

Section: Maldi Ms and Ms/msmentioning

confidence: 99%

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Zhong

Marcus

2005

J. Am. Soc. Mass Spectrom.

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148

141

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Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 g of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/ l or 37 fmol/ l. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein. (J Am Soc Mass Spectrom 2005, 16, 471-481)

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Section: Maldi Ms and Ms/msmentioning

confidence: 99%

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Zhong

Marcus

2005

J. Am. Soc. Mass Spectrom.

Self Cite

148

141

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“…Several publications claim that peptides that are more hydrophobic or more basic are more detectable (44,50). Promising results were reported when signal reproducibility was improved by using a matrix-comatrix system (21,48,51) or using a "sandwich" (52) or other improved sample preparation methodology (1,(53)(54)(55). A single component matrix and solvent-free sample separations (28,56) and ionic liquid matrix also called room temperature ionic liquids (6,7,12,27,57,58) were also used to improve the detectability of the analytes and to acquire calibration curves with good linearity and accuracy for quantitation.…”

mentioning

confidence: 99%

Investigating the Quantitative Nature of MALDI-TOF MS

Szaéjli

Feheér

Medzihradszky

2008

Molecular & Cellular Proteomics

136

110

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MALDI-TOF MS has been applied by several groups to relative quantitative measurements. At the same time, the non-quantitative character of this method has been widely reported. We conducted experiments to test the reliability of this technique for quantitation using the statistical method of inverse confidence limit calculation for the first time in this context. The relationship between relative intensities of known amounts of standard peptides and their concentration ratios was investigated. We found that the concentration ratios determined by relative intensity measurements were highly inaccurate and strongly influenced by the molecular milieu of the sample analyzed. Thus, we emphasize the necessity of using the sample itself for calibration. We also performed experiments using an isotope-labeled derivative of the analyte as an internal standard for calibration line generation. As expected, the use of such standard led to a dramatic increase in precision and a less pronounced improvement in accuracy. We recommend performing a similar statistical analysis as a demonstration of reliability for every system where MALDI-TOF MS is used for quantitative measurements. Molecular & Cellular Proteomics 7: 2410 -2418, 2008.MALDI-TOF MS is a widely used analytical technique because of its excellent sensitivity, relatively high speed, and simplicity. As a consequence of producing mostly singly charged ions (1, 2) MALDI is especially suitable for mixture analysis. It is commonly used in proteomic studies (3) frequently without any prefractionation (4) as well as in the analysis of other biomolecules. Lately MALDI-TOF MS has become a popular method for quantitative analysis of biomolecules (oligonucleotides, proteins, glycoproteins, etc.) originating from various sample types (5-22) or even for imaging tissue sections (13)(14)(15)(16)(17)(18)(23)(24)(25)(26). At the same time, several studies discuss the non-quantitative nature of MALDI-TOF MS (7, 8, 13, 19, 22, 26 -30). The main problem of MALDI is its poor reproducibility (sample to sample and shot to shot), which makes most quantitative measurements quite difficult at best (19,27,31). This is especially true for complex samples. The presence of so-called hot spots or sweet spots is the main cause of the poor reproducibility and is therefore a factor strongly increasing measurement times and complicating automated measurements. Furthermore this phenomenon is one of the major factors hampering the use of MALDI MS for spatially resolved imaging (27). The reasons for sweet spot formation are not completely understood. It could be caused by variation in the amount and the crystallization of the matrix, the latter being a function of local analyte concentration. Dependence of signal intensities on the orientation of the matrix-analyte crystals relative to the spectrometer axis is also possible (27). The extent of hot spot formation depends strongly on the matrix used. For example, preparations with ␣-cyano-4-hydroxycinnamic acid (CHCA) 1 deliver relatively homogenous samples (...

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“…There are various types of sample preparation methods for MALDI-TOF-MS analysis, such as the drieddroplet [1], fast evaporation [33], vacuum drying [34], sandwich [35], and two-layer [36] methods. Among these techniques, the dried-droplet method is one of the most frequently used for MALDI-TOF-MS analysis in the case of solution (analyte) to solution (matrix) conditions.…”

Section: Optimizing Sample Preparation Methodsmentioning

confidence: 99%

MALDI-TOF-MS analysis of small molecules using modified mesoporous material SBA-15 as assisted matrix

Kim

et al. 2009

J. Am. Soc. Mass Spectrom.

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Mesoporous silica, SBA-15 was successfully functionalized with quinoline moiety, and was applied as a matrix in the MALDI-TOF-MS analysis of small molecules. The modified SBA-15 material propoxy) quinoline, SBA-15-8QSi] was obtained by using calcined SBA-15 and 8-hydroxy quinoline. The structure of the functionalized mesoporous material was systemically characterized by TEM, the N 2 adsorption-desorption isotherm technique and FT-IR spectra. Compared with DHB and SBA-15, SBA-15-8QSi demonstrated several advantages in the analysis of small molecules with MALDI-TOF-MS, such as less background interference ions, high homogeneity, and better reproducibility. Based on these results, the various analytical parameters were optimized. The ideal operating conditions were (1) methanol used as the dissolving solvent; (2) sample first dropping method; (3) a ratio between the analyte and the matrix of 3.5:10. Under these optimization conditions, a low detection limit (8 pmol for L-Arginine-HCl) and high reproducibility (Յ29%) were obtained. This technique was successfully applied to the analysis of various types of small molecules, such as saccharides, amino acids, metabolites, and natural honey. [7], and synthetic polymers [8,9]. However, the application of MALDI-TOF-MS in small molecules analysis has been limited, mainly due to ion interference originating from matrices in the low molecule weight region (Ͻ500 Da) [10,11]. To overcome this problem, several alternative matrices, such as high mass molecules [12,13] There has been a great deal of interest in ordered mesoporous materials due to their regular pore sizes in the mesoporous range, high surface areas, high pore volumes, and easy functionalization since they were first reported in 1992 [23,24]. Recently, there have been significant advances in the preparation and modification of mesoporous materials, which allows their utilization in various applications, such as adsorbents [25], catalyst supports [26], nano-templates [27], and nanomaterials [28] for advanced electronics. In general, the pore surfaces of mesoporous silica materials are covered with a layer of silanol groups (Si-OH), which can act as the binding sites for desired functional groups via covalent grafting on the surfaces. Hu et al. reported on titanium (IV) immobilized mesoporous silica particles which were used as an enriching trap for small molecules (phosphorylated peptides), determined with MALDI-TOF-MS [29]. In addition, mesoporous silica materials have the advantage of optical transparency in the visible region [30].Herein, we successfully functionalized the mesoporous silica, SBA-15 with an 8-hydroxy quinoline group (SBA-15-8QSi hereafter), and applied the functional material as a matrix for MALDI-TOF-MS analysis. The SBA-15-8QSi material exhibited less ion backAddress reprint requests to Dr.

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Two-Layer Sample Preparation: A Method for MALDI-MS Analysis of Complex Peptide and Protein Mixtures

Cited by 201 publications

References 23 publications

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Investigating the Quantitative Nature of MALDI-TOF MS

MALDI-TOF-MS analysis of small molecules using modified mesoporous material SBA-15 as assisted matrix

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