Label-free Quantitative Proteomics Using Large Peptide Data Sets Generated by Nanoflow Liquid Chromatography and Mass Spectrometry

Abstract

We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000 -160,000 peaks above the predetermined threshold detectable in 60-g cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10

Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas

“…The 42 paired samples of N and T from the initial cohort, for which protein samples were available, were subjected to 2DICAL analysis 26. One milligram of frozen tissue was digested using trypsin with 1% sodium deoxycholate, and the digested peptides were extracted in aqueous phase according to the phase transfer surfactant protocol 27.…”

“…The extracted peptide samples were dried and dissolved in 0.1% formic acid to the final concentration of 150 ng/ml, and analyzed in duplicate by nanoflow high‐performance liquid chromatography (NanoFrontier nLC, Hitachi High‐technologies, Tokyo, Japan) connected to an electrospray ionization quadrupole time‐of‐flight mass spectrometer (TripleTof 5600, AB Sciex, Framingham, MA). Mass spectrum peaks were detected, normalized, and quantified using our 2DICAL software package 26. If the ratio of the expression level of T to the corresponding N ( P T/N ) was 2 or more, the protein expression of the gene was considered to be elevated in the T sample relative to the N sample.…”

Label-free quantitation: A new glycoproteomics approach

“…To alleviate much of the variation due to changes in MS response among samples, normalization of data has been applied in proteomics studies [33][34][35][36]. Many proteomics researchers employ a normalization technique that involves dividing intensities of individual peaks by the total intensity from all peaks in the spectrum [33][34][35]; this approach is fundamentally similar to the approach by Wang et al, where spectra are normalized by calculating an intensity ratio for one spectrum versus another by doing pair-wise comparisons of peak intensities, for all the peaks in the spectra [36].…”

“…Many proteomics researchers employ a normalization technique that involves dividing intensities of individual peaks by the total intensity from all peaks in the spectrum [33][34][35]; this approach is fundamentally similar to the approach by Wang et al, where spectra are normalized by calculating an intensity ratio for one spectrum versus another by doing pair-wise comparisons of peak intensities, for all the peaks in the spectra [36]. In the work presented here, this concept of normalization is builtupon to produce a label-free quantitative method for glycopeptide analysis.…”

“…In the work presented here, this concept of normalization is builtupon to produce a label-free quantitative method for glycopeptide analysis. While in the proteomic field one can obtain reliably quantitative data by normalizing the data to the total ion abundance [33][34][35], this method is potentially problematic for glycopeptide analysis because the glycopeptides ionize weakly, compared with the nonglycosylated peptides that may also be present. Therefore, small changes in the presence of nonglycosylated interferents could impart large variability in a quantitative assay when the total ion current (or pairwise comparison of all the peaks) is used to normalize the ion abundances of the analytes.…”