Lex mixtures of proteins. Application for evaluation of picogram array of RAW 264.7 cell lysate The integrated program was used to analyze RAW 264.7 cell lysate in triplicate. Two buffers had been applied to prepare the protein samples. In a single case, a sample containing 0.1 mg/mL proteins was dissolved in 50 (v/v) ACN, 0.025 (w/v) SDC, and five mM NH4HCO3. Within a second case, a 1 mg/mL protein sample was dissolved in 50 (v/v) ACN, 0.25 (w/v) SDC, and 5 mM NH4HCO3. The injection amounts per run for the 0.1 mg/mL and 1 mg/mL samples have been 300 pg and 3 ng, respectively. Just after triplicate analysis, two 1 and 7 2 protein groups had been identified in the 300 pg and 3 ng RAW 264.7 cell lysates, respectively. Information with the identified protein groups is listed in supporting material I. The protein groups (except 1 protein group, Glial fibrillary acidic protein) obtained from the 300 pg sample were also identified from the 3 ng sample. For glial fibrillary acidic protein, one particular exceptional peptide was identified, and its Exp worth and mass error have been 1.4E-06 and 1 ppm, respectively, which confirmed the protein group identification. The majority of the proteins identified from the 300 pg and 3 ng samples have been comparatively higher abundant in RAW 264.7 cell lysate in accordance with the protein spectral count info in the large-scale proteome data generated previously in our group. Moreover, the peptide identifications in the 300 pg and three ng samples have been also manually evaluated, along with the annotated tandem spectra of those peptides are listed in supporting material II. We also analyzed the molecular weight and pI of proteins generated by triplicate analysis from the 3 ng RAW 264.7 cell lysate. The molecular weight ranged from 5.7 kDa to 58.8 kDa, and pI ranged from four.five to 11.0, which recommend that the integrated CZE-ESI-MS/NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAnal Chem.Bezuclastinib Author manuscript; offered in PMC 2014 April 16.Sun et al.PageMS program is effective for modest and massive proteins, and also acidic and simple proteins. We further checked the disulfide bonds with the identified proteins from three ng RAW 264.7 cell lysate, and the identified proteins are likely to have handful of or no disulfide bonds, that is expected because of the a lot simpler denaturation of these proteins with 50 (v/v) ACN. The outcome indicates that the integrated CZE-ESI-MS/MS system may well have potential limitations for identification of hugely structured proteins with several disulfide bonds. On the other hand, the program might be valuable for selective identification of your proteins with handful of disulfide bonds in complex samples.Epratuzumab We additional extracted the electropherograms from a peptide from macrophage migration inhibitory factor, which was identified from each 300 pg and 3 ng samples, Fig.PMID:23672196 4. The integrated CZE-ESI-MS/MS method yielded reasonably reproducible migration time for the peptide for analysis of both 300 pg and three ng samples. Also, the peptide intensity from triplicate analysis in the 3 ng cell lysate was fairly consistent (RSD = eight ), as well as the peptide intensity from the 300 pg cell lysate was reasonably reproducible (RSD = 30 ). Furthermore, when the injection amount was elevated from 300 pg to 3 ng, the average peptide intensity generated by the system increased from 7.5E+04 to 7.6E+05. The reproducible peptide migration time and peptide intensity indicate that the integrated CZE-ESI-MS/MS program is reproducible for trace complicated cell lysate evaluation. The peptide intensities ge.
