Mass spectrometry peptide analysis

Medzihradszky, K.F., H. Leffler, M.A. Baldwin and A. Burlinghame. Protein identification by in-gel digestion, high-performance liquid chromatography and mass spectrometry peptide analysis by complementary ionization techniques. J. Am. Soc. Mass Spectrom. 12 215-221, 2001. 

A. J. Tomlinson and S. Naylor, Systematic development of on-line membrane preconcentration-capillary electrophoresis-mass spectrometry foi analysis of peptide mixtures, J. Capillary Electrophoresis 225-233 (1995). 

Liang, Z., Duan, J., Zhang, L., Zhang, W., Zhang, Y., and Yan, C. (2004). Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins. Anal. Chem. 76, 6935-6940. 

The methods for each study are divided into the initial protein separation step, a second separation step if applicable, the type of mass analysis, and the software used for peptide identification. ID = one dimensional polyacrylamide gel electrophoresis, 2D = two dimensional polyacrylamide gel electrophoresis, MS = mass spectrometry (peptide mass fingerprinting), MS/MS = tandem mass spectrometry, MALDI-TOF = matrix assisted laser desorption/ionization-time of flight, MS FIT = software from Protein Prospector, http //prospector.ucsf edu/, ESI = electrospray ionization, Q-TOF = quadrupole-time of flight, PPSS2 =Protana s Proteomic Software Suite (ProtanaEngineering, Odense, Denmark), Mascot = Matiix Science, http //www.matrixscience.com/, TOF-TOF = MALDI plus TOF tandem mass spectrometry, RP-HPLC = reverse phase high performance liquid chromatography, Q-IT = quadrupole ion trap, LIT = linear ion trap. Bioworks = Thermo Electron Corporation. 

Electrospray ionization and MALDI represent major advances in the application of mass spectrometry to analysis of biomolecules. Although in many cases the two techniques are able to provide the same information, quite frequently they are complementary. For a variety of reasons, some samples are more successfully analyzed by MALDI and other by ESI. Thus, it is highly beneficial to have access to both types of instrument for characterization of synthetic peptides. In ESI-MS, the samples are introduced in solution at flow rates from less than 1 tL/min up to 1 mL/min, depending on the design of the ESI interface. Pure samples and simple mixtures are often analyzed by direct infusion more complex mixtures (such as proteolytic digests) generally require either on-line or off-line HPLC fractionation prior to MS analysis. 

Figure 1.2 The classical continuous labeling (bottom-up) HX-MS experiment. HX of an equilibrated protein solution is initiated by dilution into a Dfl-containing buffer, and exchange is quenched at various time points. Global HX (protein level) can be measured directly by liquid chromatography (LC) and mass spectrometry (MS) analysis of the intact protein, or local HX (peptide level) can be measured by enzymatic cleavage and subsequent LC-MS analysis of the proteolytic peptides. (See insert for color representation of the figure.)...

Traditional methods to generate peptide maps involve fractionation of complex mixtures of peptides in a protein digest either with one-dimensional SDS-PAGE or RP-HPLC [28,29]. The mass spectrometry peptide-mapping protocol, in principle, is similar to these techniques, but it provides an added dimension of structure-specific data (i.e., the molecular mass). MALDI-MS [30,31], ESl-MS [32], LC/ESI-MS [33], and CE/ESI-MS [34] have currently replaced the traditional biochemical approaches. MALDI allows the direct analysis of unfractionated protein digests. The commonly used matrices are sinapinic acid, a-cyano-4-hydroxy cinnamic acid (a-CHCA), and 2,5-dihydroxybenzoic acid (DHB). 

Oven/iew Waters, Sediments, and Soils. Ion-Selective Electrodes Water Applications. Isotope Dilution Analysis. Liquid Chromatography Size-Exclusion Liquid Chromatography-Mass Spectrometry Mass Spectrometry Peptides and Proteins. Voltammetry Overview. 

See alsa Chromatography Multidimensional Techniques. Environmental Analysis. Extraction Solid-Phase Extraction. Food and Nutritional Analysis Sample Preparation Contaminants Pesticide Residues. Forensic Sciences Drug Screening in Sport Illicit Drugs. Herbicides. Liquid Chromatography Instrumentation Clinical Applications Food Applications. Mass Spectrometry Peptides and Proteins. Pesticides. Pharmaceutical Analysis Sample Preparation. Proteomics. Sample Handling Automated Sample Preparation. Water Analysis Organic Compounds. 

See also Capillary Electrophoresis Overview. Chir-optical Analysis. Liquid Chromatography Column Technology Mobile Phase Selection Reversed Phase Instrumentation Amino Acids. Mass Spectrometry Peptides and Proteins. Nuclear Magnetic Resonance Spectroscopy Techniques Nuclear Overhauser Effect. Proteins Traditional Methods of Sequence Determination Foods. 

The different stages of the preparation of peptide surfaces can be confirmed with surface-sensitive physical and chemical analysis techniqnes. For gold-based SAMs, Mrksich and co-workers have introduced a matrix-assisted laser desorption ionisation time-of-ftight (MALDI-TOF) mass spectrometry-based analysis procedure with which they are able to identify the presence of various surface functional groups via their mass (Yeo Mrksich, 2006 Yeo et al., 2003). Although this method is applicable to SAMs, it is not strictly a surface sensitive technique, as the desorption process in MALDI is not confined to the uppermost layer of a material. 

Schmidt A-C, Fahlbusch B, Otto M. Size exclusion chromatography coupled to electrospray ionization mass spectrometry for analysis and quantitative characterization of arsenic interactions with peptides and proteins. J Mass Spectrom 2009 44 898-910. 

The first reports of metabolic labeling appeared in 1999. In this procedure, yeast cells were grown in two media, one of which used N-emiehed media. " The two yeast cultures were combined and the proteins of interest were digested with trypsin before mass speetrometry analysis. The labeling by use of an N-ammonium salt results in the eomplete labeling of amino acid and could be quantified with mass spectrometry. The corresponding mass shift between the unlabeled and the labeled form of the peptide requires high resolution mass spectrometry for analysis. 

Until 1981, mass spectrometry was limited, generally, to the analysis of volatile, relatively low-molecular-mass samples and was difficult to apply to nonvolatile peptides and proteins without first cutting them chemically into smaller volatile segments. During the past decade, the situation has changed radically with the advent of new ionization techniques and the development of tandem mass spectrometry. Now, the mass spectrometer has a well-deserved place in any laboratory interested in the analysis of peptides and proteins. 

The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of 10 kDa, the sensitivity of FAB is quite low, and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. 

Chapter 40 Analysis of Peptides and Proteins by Mass Spectrometry... 

The use of mass spectrometry for the analysis of peptides, proteins, and enzymes has been summarized. This chapter should be read in conjunction with others, including Chapter 45, An Introduction to Biotechnology, and Chapters 1 through 5, which describe specific ionization techniques in detail. 

Table 5.5 Nomenclature of the ions formed in the mass spectral fragmentation of polypeptides. From Chapman, J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. Reproduced by permission of Humana Press, Inc. 

J. R. (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Methods in Molecular Biology, Vol. 61, 1996. 

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