Protein mass, determination

Technologies that offer the opportunity to use MS for intact protein mass determination allow rapid assessment of signal peptide cleavage sites and post-translational heterogeneity. A well-resolved mass spectrum of an intact protein defines its native covalent profile as well as its associated heterogeneity. A comprehensive mass spectrometric approach that integrates both intact protein molecular mass measurement (top-down) and proteolytic fragment identification (bottom-up) would represent a powerful approach for proteome/ protein characterization. 

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. 

A biochemist isolates a new protein and determines its molar mass by osmotic pressure measurements. A 50.0-mL solution is prepared by dissolving 225 mg of the protein in water. The solution has an osmotic pressure of 4.18 mm Hg at 25°C. What is the molar mass of the new protein ... 

Electrospray ionization mass spectrometry (ESI-MS) is an analytical method for mass determination of ionized molecules. It is a commonly used method for soft ionization of peptides and proteins in quadmpole, ion-trap, or time-of-flight mass spectrometers. The ionization is performed by application of a high voltage to a stream of liquid emitted from a capillaty. The highly charged droplets are shrunk and the resulting peptide or protein ions are sampled and separated by the mass spectrometer. 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

Figure 2.4. Peptide fingerprinting by MALDI-TOF mass Spectrometry. Proteins are extracted and separated on by 2D gel electrophoresis. A spot of interest is excised from the gel, digested with trypsin, and ionized by MALDI. The precise mass of proteolytic fragments is determined by time-of- flight mass spectrometry. The identity of the protein is determined by comparing the peptide masses with a list of peptide masses generated by a simulated digestion of all of the open reading frames of the organism.

Since ProteinTrawler records the retention time of each protein mass, it is a simple endeavor to maintain chromatographic conditions, split the flow that exits the LC column with a small portion set to the mass spectrometer to monitor for assurance that there were no changes in the retention time that would hinder the pooling of fractions from multiple runs, and to facilitate the determination of which fractions contained the desired proteins. In our experimental setup, the flow was split after the column with 25% of the flow going to the mass spectrometer while the remaining diverted to an HP1100 fraction collector. The fraction collector was used to collect fractions at 1.0-minute intervals. 

Li, Y. Mclver, R. T., Jr. Hunter, R. L. High-accuracy molecular mass determination for peptides and proteins by Fourier transform mass spectrometry. Anal. Chem. 1994, 66, 2077-2083. 

Because online separations provide such a wealth of information about target proteins, interpretation becomes of critical importance in order to make full use of the data. The first step in any analysis of LC-MS data involves integration and deconvolution of sample spectra to determine protein mass and intensity. In manual analysis (Hamler et al., 2004), users identify protein umbrellas, create a total ion chromatogram (TIC), integrate the protein peak, and deconvolute the resulting spectrum. Deconvolution of ESI spectra employs a maximum entropy deconvolution algorithm often referred to as MaxEnt (Ferrige et al., 1991). MaxEnt calculates... 

Although most assays perform well with regard to specificity and reproducibility, the major problem remains their standardization (A9, Dl, K30, L4). There is currently no internationally accepted standard, and the selection of a reference material raised many problems (A8, G5, K30, L4). A number of questions have not been solved Should the standard consist of several apo(a) isoforms Can the reference material be lyophilized Should results be expressed as mass or as moles of apoprotein or lipoprotein How should the protein mass of the primary standard be determined What are optimal storage conditions for the secondary standard Which method can be used as a reference method Can recombinant apo(a) represent an alternative for a primary standard These problems came to light in the course of the international surveys whose results were presented at the Lp(a) Workshop in New Orleans (1992) (L4). 

Reports exact molecular weight of a protein or part of a protein as determined by mass spectrometric methods Any comment that does not belong to any of the other defined... 

Dobberstein, P. Schroeder, E. Accurate Mass Determination of a High Molecular Weight Protein Using ESI With a Magnetic Sector Instrument. Rapid Commun. Mass Spectrom. 1993, 7, 861-864. 

Mass spectrometry studies on proteins can determine the purity of the sample, verify amino acid substitutions in mutants, detect post-translational modihcations, or calculate the number of disulfide bridges. Amino acid... 

The relative molecular mass determination of an unknown protein is generally performed automatically using various deconvolution algorithms, but the procedure is limited to relatively simple mixtures. 

Figure 1. Top Turbidity, measured at 350 nm, as a function of microtubule polymer mass concentration (expressed as mg/mL polymerized tubulin). Tubulin solutions of varying concentrations were polymerized until they reached stable plateau values in a Cary 118C spectrophotometer. Each sample was then transferred to an ultracentrifuge tube, and microtubules were pelleted, separated from the unpolymerized tubulin in the supernatant fraction, and then resuspended for protein concentration determination. The corresponding turbidity and polymer mass concentrations are plotted here. Bottom Time-course of tubulin polymerization assayed by turbidity.Repro-duced from MacNeal and Purich with permission from the American Society for Biochemistry and Molecular Biology.

Selected entries from Methods in Enzymology [vol, page(s)] Association constant determination, 259, 444-445 buoyant mass determination, 259, 432-433, 438, 441, 443, 444 cell handling, 259, 436-437 centerpiece selection, 259, 433-434, 436 centrifuge operation, 259, 437-438 concentration distribution, 259, 431 equilibration time, estimation, 259, 438-439 molecular weight calculation, 259, 431-432, 444 nonlinear least-squares analysis of primary data, 259, 449-451 oligomerization state of proteins [determination, 259, 439-441, 443 heterogeneous association, 259, 447-448 reversibility of association, 259, 445-447] optical systems, 259, 434-435 protein denaturants, 259, 439-440 retroviral protease, analysis, 241, 123-124 sample preparation, 259, 435-436 second virial coefficient [determination, 259, 443, 448-449 nonideality contribution, 259, 448-449] sensitivity, 259, 427 stoichiometry of reaction, determination, 259, 444-445 terms and symbols, 259, 429-431 thermodynamic parameter determination, 259, 427, 443-444, 449-451. 

Molar mass determinations based on SDS-PAGE is sometimes misleading, since some proteins are not conversed completely into a rod-like shape or the protein/SDS ratio differs from the average. 

For characterization of a GPC medium other than defined macromolecules as dextrans, polypeptides, polystyrene sulfates, or colloidal gold may be used. For these standards the eluent is ddH20. These macromolecules are suitable for molar mass determination of proteins with restrictions only. 

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