Mass spectrometry: A tool for the identification of proteins separated by gels

Gold nanoparticles (AuNPs) have been studied as a potential solid-state matrix for laser desorption/ionization mass spectrometry (LDI-MS) but the efficiency in ionization remains low. In this report, AuNPs are capped by a self-assembled monolayer of cysteamine and modified with ␣-cyano-4-hydroxycinnanic acid (CHCA) for effective MALDI measurements. CHCA-terminated AuNPs offer marked improvement on peptide ionization compared with citrate-capped or cysteamine-capped AuNPs. The coating also effectively suppresses formation of Au cluster ions and analyte fragment ions, leading to cleaner mass spectra. Addition of glycerol and citric acid to the peptide/AuNPs sample further improves the performance of these AuNPs for LDI-MS analysis. Glycerol appears to enhance the dispersion of AuNPs in sample spots, increasing the sample ionization and shot-to-shot reproducibility, while citric acid serves as an external proton donor, providing high production of protonated analyte ions and reducing fragmentation of peptides on the nanoparticle-based surface. Optimal ratios of citric acid, glycerol, and AuNPs in sample solution have been systematically studied. A more than 10-fold increase for desorption ionization of peptides can be achieved by combining 5% glycerol and 20 mM citric acid with the CHCA-terminated AuNPs. The applicability of the CHCA-AuNPs for LDI-MS analysis of protein digests has also been demonstrated. This work shows the potential of AuNPs for SALDI-MS analysis, and the improvement with chemical functionalization, controlled dispersion, and use of an effective proton donor. (J Am Soc Mass

mentioning

confidence: 99%

CHCA-modified Au nanoparticles for laser desorption ionization mass spectrometric analysis of peptides

Duan

Linman

Chen

et al. 2009

“…Especially in the field of proteomics, these methods have proven to be very powerful both for the identification of known proteins and for the sequencing of unknown proteins and peptides. The metastable decay of preselected parent ions in MALDI has also proven to be very useful in proteomics [1][2][3], and is today a key technique in sequence analysis in proteomics. However, at the same time, the performance of these methods has not met expectations when it comes to the analyses of DNA segments.…”

mentioning

confidence: 99%

Sodium controlled selective reactivity of protonated deoxy-oligonucleotides in the gas phase

Flosadóttir

Staňo

Ingólfsson

2008

Metastable fragmentation of the positively charged, hexameric oligonucleotides 5=-d(TTXYTT) (X and Y are dC, dG, or dA) and 5=-d(CTCGTT), 5=-d(TTCGTC) and 5=-d(CTCGTC) is studied after matrix assisted laser desorption/ionization (MALDI). The influence of the degree of sodiation, i.e., when the acidic protons are one by one exchanged against sodium ions, is systematically studied for the exchange of up to seven protons against sodium ions. Exchanging the acidic protons against sodium gradually quenches the backbone cleavage through the w and a-B channels, and quantitative quenching of these channels is generally achieved with the exchange of four protons against sodium ions. At the same time, the exchange of protons against sodium ions promotes the loss of a neutral, high proton affinity base. The formation of the w and a-B fragments is found to be highly dependent on the sequence of the central bases. A single mechanism consistent with these observations is proposed. In addition to the quenching of the classical w and a-B reaction channels, a drastic and abrupt on/off-switching of new reaction channels is observed as the degree of sodiation successively increases. These channels involve selective loss of the two central bases and the excision of a phosphodiester group and a sugar unit from the center of the oligonucleotides. Synchronously, the two terminal fragments recombine to form a tetramer containing the two terminal nucleosides from each end of the hexamer. Possible mechanism explaining these remarkable channels are discussed. T he need for new analytical methods and mass spectrometric techniques to shed light on the complex molecular structure and composition of biologically relevant molecules has drawn the attention of physicists and physical chemists more in the direction of gas-phase studies on molecules such as DNA, RNA, proteins, and peptides. The introduction of the electrospray ionization (ESI) and the matrix-assisted laser desorption ionization (MALDI) in the early 90s and the further development of these methods throughout the last decade have revolutionized the studies of biologically relevant molecules. Especially in the field of proteomics, these methods have proven to be very powerful both for the identification of known proteins and for the sequencing of unknown proteins and peptides. The metastable decay of preselected parent ions in MALDI has also proven to be very useful in proteomics [1][2][3], and is today a key technique in sequence analysis in proteomics. However, at the same time, the performance of these methods has not met expectations when it comes to the analyses of DNA segments. One of the difficulties hampering the use of MALDI TOF-MS in DNA analyses is the metastable and prompt decay of these molecules in the MALDI process [4 -11]. Furthermore, the strong affinity of oligonucleotides to exchange their numerous acidic protons against sodium complicates the sample preparation considerably and often sets limits to the achievable resolution [4,5]. In the last decades, several stud...

“…MALDI-MS peptide mass fingerprinting of isolated proteins with peptides derived from endoproteolytic digests has been widely established as the technique of choice for high throughput protein identification [1][2][3]. After annotation and calibration of mass spectrometric data, the observed peptide masses are compared with molecular weights derived from an in-silico digest of a sequence database.…”

mentioning

confidence: 99%

Algorithm for accurate similarity measurements of peptide mass fingerprints and its application

Monigatti

Berndt

2005

We present a simple algorithm which allows accurate estimates of the similarity between peptide fingerprint mass spectra from matrix assisted laser desorption/ionization (MALDI) spectrometers. The algorithm, which is a combination of mass correlation and intensity rank correlation, was used to cluster similar spectra and to generate consensus spectra from a data store of more than 100,000 spectra. The resulting first spectra library of 1248 unambiguously identified different protein digests was used to search for missed cleavage patterns that have not been reported so far and to shed light on some peptide ionization characteristics. The findings of this study could be directly implemented in peptide mass fingerprint search algorithms to decrease the false positive error rate to Ͻ0.25%. Furthermore, the results contribute to the understanding of the peptide ionization process in MALDI experiments. (J Am Soc Mass Spectrom 2005, 16, 13-21)