Electrospray ionization mass spectrometry intact proteins

Whitelegge, J. P. Gundersen, C. B. Faull, K. F. 1998. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins. Protein Sci., 7,1423-1430. 

Balaguer, E. and Neusuess, C. (2006) Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry. Anal Chem, 78 (15), 5384-5393. 

The introduction and eventual commercialization of matrix-assisted laser desorption/ionization (MALDI) and electrospray (ESI) allowed biomarker status to be extended to proteins in 1996.15"17 With a few exceptions, ESI has been used in conjunction with extractions and high-pressure liquid chromatography (UPLC) interfaced with mass spectrometry. MALDI, on the other hand, has been widely adapted for rapid analysis of intact organisms, supported by bioinformatics.1819... 

Liu, H. J., Berger, S. J., Chakrahorty, A. B., Plumh, R. S., Cohen, S. A. (2002). Multidimensional chromatography coupled to electrospray ionization time-of-fhght mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins. J. Chromatogr. B 782(1-2), 267-289. 

J. A. Loo, C. G. Edmonds, and R. D. Smith. Primary Sequence Information from Intact Proteins by Electrospray Ionization Tandem Mass Spectrometry. Science, 248(1990) 201-204. 

Mass spectrometry (MS) is widely used to ascertain the purity, total mass of the protein produced, and detect any covalent modifications (Cohen and Chait, 2001). Both electrospray ionization (ESI) and MALDI may be used although for intact proteins ESI has the advantage of being accurate to 1 Da. Using the simple protocol described in Protocol 2.11, the MS of whole protein samples can be readily automated without the need for sample preparation. This method has proved successful for the... 

Electrospray mass spectrometry utilizes a soft ionization technique at nearly atmospheric pressure. As a result, intact molecular ions are formed in high yield. The instrument can be interfaced readily to HPLC or capillary electrophoresis columns and sub-femtomole amounts of proteins can be detected. A disadvantage is that salt concentrations must be kept low (< mM) and that the protein tends to bind Na, K+, and anions that may confuse interpretation of spectra. 

Coupling mass spectrometry (MS) to capillary electrophoresis provides detection and identihcation of amino acids, peptides, and proteins based on the accurate determination of their molecular masses [15]. The most critical part of coupling MS to CE is the interface technique employed to transfer the sample components from the CE capillary column into the vacuum of the MS. Electrospray ionization (ESI) is the dominant interface which allows a direct coupling under atmospheric pressure conditions. Another distinguishing features of this soft ionization technique when applied to the analysis of peptides and proteins is the generation of a series of multiple charged, intact ions. 

Newer developments in this area combine analytical techniques to achieve a 2-D separation by linking, for example, liquid lEF with nonporous silica reverse phase, high performance liquid chromatography (HPLC see Chapter 6) and detecting intact proteins by electrospray ionization, time of flight, and mass spectrometry (see Chapter 7). 

Li, W. Hendrickson, C.L. Emmett, M.R. Marshall, A.G. Identification of Intact Proteins in Mixtures by Alternated Capillary Liquid Chromatography Electrospray Ionization and LC ESI Infrared Multiphoton Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Anal. Chem. 71, 4397 402 (1999). 

Williams, D.K. Hawkiidge, A.M. Muddiman, D.C. Sub-ppm mass measurement accuracy of intact proteins and product ions achieved using a dual electrospray ionisation quadrupole FT-ICR mass spectrometer. J. Am. Soc. Mass Spectrom. 2007,18 ), 1-7. Witt, M. Fuchser, J. Baykut, G. FT-ICR mass spectrometry with NanoLC/microelec-trospray ionization and MALDI Analytical performance in peptide mass fingerprint analysis. /. Am. Soc. Mass Spectrom. 2003,14(6), 553-561. 

Intact protein mass spectrometry allows the molecular mass determination of either proteins or complexes of proteins and covalently bound ligands/other proteins. In a first step, the sample is desalted to detach from buffer components and small ions that would interfere through noncovalent complexes in the gas phase. Next, the isolated protein is ionized, for example, by electrospray ionization (ESI). The acid in the eluent causes protonation of the protein at basic sites, particularly lysine and arginine residues, so that m/z values of multiple species with different charges can be measured in a mass spectrometer. These data are then combined during the deconvolution process to yield the mass of the protein or complex. 

Top-down proteomics [1] is the identification and characterization of a mature, intact protein (or proteins) using primarily mass spectrometry (MS)-based techniques and the sequence information contained in genomic/proteomic databases. Unhke bottom-up proteomics [2-4], where a protein (or proteins) is digested into peptides prior to MS or tandem mass spectrometry (MS/MS) analysis [3,4], the top-down approach involves measuring the molecular weight (MW) of the intact, mature protein with its associated posttranslational modifications (PTMs) if any. The intact protein ion is then fragmented in the gas phase in order to determine its amino acid sequence as well as the location and identification of PTMs. From its earliest development, top-down proteomics has been primarily the domain of electrospray ionization (ESI) [5] (which generates mul-... 

Since the development of electrospray ionization and MALDI, large scale application of mass spectrometry to study of proteins has become possible. Broadly, two different approaches have been followed to characterize proteins. In the first, intact proteins ire ionized by using either electrospray or MALDI. Ionized proteins are then introduced into the mass analyzer. Alternatively, proteins are first digested into smaller peptides using different proteases and a collection of these smedler peptides is then introduced into the mass analyzer. The latter technique is preferred. 

Loo, J. A. Edmonds, C. G. Smith, R. D. Primary sequence information from intact proteins by electrospray ionization tandem mass spectrometry. Science 1990, 248, 201-204. 

---