Protein identity

Isobaric labels thus permit quantitative information regarding protein expression levels in multiple samples analyzed simultaneously by MS. The multiplexed capability of these reagents allows the measurement of peptides and proteins in diseased samples, treated samples, and normal samples all in the same experiment. In addition, since all peptides from a given protein get labeled at their N-termini, the MS analysis generates more than one peptide signal, which can be used to confirm protein identity with greater confidence than using a cysteine label, like ICAT. 

X 96) proteins simultaneously for automatic analysis (Houthaeve etal., 1997 Jensen etal., 1997). In clinical applications this method could be used to screen complete 2D gels and annotate them automatically with the protein identity (Hoogland et al., 1998). 

S. Medda, K. Takeuchi, D. Devore-Carter, O. von Diemling, E. Heymann, R. T. Swank, An Accessory Protein Identical to Mouse Egasyn Is Complexed with Rat Microsomal /1-Glucuronidase and Is Identical to Rat Esterase-3 ,. /. Biol. Chem. 1987, 262, 7248-7253. 

The primary analytical applications of RPLC in the development of biopharmaceuticals are the determination of protein purity and protein identity. Purity is established by analysis of the intact protein, and RPLC is useful in detecting the presence of protein variants, degradation products, and contaminants. Protein identity is most often established by cleavage of the protein with a site-specific protease followed by resolution of the cleavage products by RPLC. This technique, termed peptide mapping, should yield a unique pattern of product peptides for a protein that is homogeneous with respect to primary sequence. 

Following the separation process, the individual proteins must be identified and characterized. The 2-D separation has provided Mr, pi, and relative abundance data but no information on protein identities or functions. Computer algorithms are available for matching the Mr, pi, and relative abundance data... 

Smalheiser, N.R., and Kim, E., 1995, Purification of cranin, a laminin binding membrane protein. Identity with dystroglycan and reassessment of its carbohydrate moieties, J Biol Chem, 270, pp 15425-15433. 

The high sample demands and low-throughput of LC-MS methods have led to the creation of a capillary electrophoresis (CE) platform for ABPP [48]. Proteomes are labeled with a fluorescent probe, digested with trypsin, and enriched with antifluorophore antibody resins. Use of CE coupled with laser-induced fluorescence (LIF) detection to analyze the enriched peptides resulted in far superior resolution to ID SDS-PAGE, particularly for enzymes that share similar molecular masses. Sensitivity limits of 0.05-0.1 pmol/mg proteome, negligible sample requirements (—0.01—0.1 pg proteome), and the ability to perform rapid CE runs in parallel with 96-channel instruments, make CE-based ABPP a potentially powerful technique. One drawback is that the identities of the probe-labeled proteins are not immediately apparent, and correlated LC-MS experiments must be performed to assign protein identities to the peaks on the CE readout. 

Peptide mapping is a method that enables the determination of protein identity when compared to a standard. When compared to previous lots of the same product, it serves to determine the stability of the protein s primary sequence, which in turn reflects the genetic stability of the producer cells. This method is capable of detecting small differences between proteins in one or more amino acids. The detection will be dependent upon an amino acid alteration affecting the observed peptide profile (Figure 13.1 see color section). 

As can be inferred, electrophoretic mobility depends on solution ionic strength since double layer thickness decreases with increasing electrolyte concentration. It also depends on the surface charge of the particles. If this charge varies in colloidal particles of similar dimensions then electrophoresis provides a basis for their separation. An example of this is in proteins, where the surface charge varies with pH in a different way according to the protein identity. 

Cold Spring Hath Symp Quant Biol LXIV 533-540 Inoue J, Kerr LD, Kakizuka A, Verma IM (1992) I kappa B gamma, a 70 kd protein identical to the C-terminal half of pi 10 NF-kappa B a new member of the I kappa B family. Cell 68 1109-1120... 

Grp78 A glucose-regulated protein identical with BiP... 

Matrix-assisted laser desorption ionization (MALDI), like ESI, is capable of ionizing and launching very large molecules (e.g, polysaccharides, synthetic polymers, peptides, and proteins) into the gas phase and is a major analytical tool for high-throughput proteomic studies.17,53 In many respects, MALDI is a complementary technique to ESI and both techniques are often applied to the same sample when determining protein identity. ESI produces macromolecular ions from solution, whereas MALDI produces them from the solid state. 

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