Next, Mass Frontier 4 was used to predict the tandem mass spectra of the candidates, which were matched to the measured spectrum, counting the number of common peaks.
Other methods, based on spectrum counting [23], are limited in their ability to detect low abundance proteins.
The rationale was that this index may suffer from biases, specifically in the spectrum counting, due to the fact that small proteins tend to have fewer peptides identified per protein compared with large proteins [ 23].
Yet a "startling fact" emerged: four of ten of those who qualified as conservatives on the ideological spectrum counted as liberal on the operational spectrum.
Differential expression of plasma proteins was estimated by comparing spectrum counts [12], [13], and significant differences determined by Mann-Whitney U test using Statview software (SAS Inc, Cary, NC, USA).
We used normalized spectrum counts to perform relative quantification and identify age- and SPARC-related changes (Table 1).
Spectral counting entails averaging the spectrum counts across the different protein samples tested, followed by calculating the quantitative value for each identified protein.
Then, nonglycosylated-peptides were released from the solid-phase of by trypsin digestion, separated by two-dimensional liquid chromatography, and analyzed by tandem MS. Protein quantification was achieved by a label-free quantitative method, which compared the spectrum counts of identified nonglycosylated-peptides between different samples.
In the next step, we collapsed redundant protein database entries (often resulting from multiple protein isoforms that were not distinguished by the peptides identified by LC-MS/MS) into single entries by adding their spectrum counts both in the mock and NKX3.1 purifications.
The mass spectra (counts versus mass/charge values for m/z 100 to 700) were exported to Excel 2007 (Microsoft Inc., Belleview, WA, USA) for data pre-processing and then to JMP 9.0 (division of SAS Institute Inc ,Cary, North Carolina, USA) to perform artificial neural network (ANN) analysis (Onofrejová et al. [2007]; Korel and Balaban [2008]).
In addition, the PfPMT, a key enzyme in the SDPM pathway as well as the S-adenosylmethionine synthetase (PFI1090w, which provides the PfPMT essential cofactor S-adenosylmethionine), showed an increase in treated versus untreated spectral counts (17 versus 5 spectra counts for PfPMT and 7 versus 2 spectral counts for PFI1090w in T4 treated and untreated parasites, respectively).
