Data Analysis

Database Searching, Mining, and Pathway Analysis

Mass spectrometry-based proteomics and metabolomics experiments generate large data sets that can be effectively exploited only with the help of appropriate software. Both commercial and internet freeware packages are available, and the Core uses and advises on both types. Proteomics experiments typically involve digestion of a protein mixture followed by LC/MS/MS analyses that generate many hundreds of peptide mass spectra often of varying quality. Mascot, Spectrum MIll, and X!Tandem will compare these spectra with theoretical spectra based on known protein sequences and generate lists of potential protein identies. Scaffold incorporates and manipulates the results of these searches and provides an efficient way to evaluate the data and provide some statistical analyses. After analysis with Scaffold, the results can be examined anywhere on a free download of Scaffold Viewer.  Metabolomics experiments often proceed in three stages; 1. initial analysis with a high-resolution instrument (ESI-TOF or QTOF) to provide exact masses, 2. comparison of results between or among various experimental conditions, 3. targeted MS/MS analysis of the compounds that changed as a result of the experiment. Significant proteins and metabolites can then be subjected to pathway analysis to gain some insight into the biochemistry of the systems; both the comparisons and the pathway analyses can be done with Mass Profiler Professional. We also have a customizable Python routine that can prepare unconventionally-modified proteins for successful Mascot searches.

Some online resources:

Quantitation: Spectral Counting, iTRAQ, and Multiple Reaction Monitoring

The Core has a long-standing focus on quantitative analysis, typically with selected-ion monitoring (SIM) or multiple-reaction monitoring (MRM) for small molecules or targeted peptides, and spectral counting or iTRAQ for proteins. It's also possible to simply integrate extracted-ion chromatograms (EIC) from non-targeted LC/MS experiments. EIC is fine for routine analyses where sensitivity is not an issue. These experiments can often be carried out on user-friendly single-quadrupole or electrospray time-of-flight instruments, and integration is done on the workstation software. If sensitivity is important, a tandem quadrupole, often called a triple-quadrupole, is the instrument of choice. For MRM experiments, an ion of interest is selected in the first quadrupole, fragmented in the second quadrupole, and then a fragment ion of choice is selected in the third. The two filters for chemical noise (in Q1 and Q3) usually result in a significant gain in signal-to-noise and consequent lower detection limits. Spectral counting is a label-free method for relative quatitation of proteins and is based on the number of spectra observed for peptides from given proteins after (usually) tryptic digestion. iTRAQ is based on isotopically-labeled tags with the same total mass that fragment in Q2 to give reporter ions with different masses. iTRAQ is thus potentially ideal for exeriments involving comparisons of several different conditions or time points. Data from these experiments are analyzed by Spectrum Mill, Scaffold, and Scaffold Q+. Check out some of the links below before contacting us via the online inquiry form to discuss the best approach for your project.

Online Resources:

Routine Statistics

GraphPad Prism is useful for curve-fitting and standard statistical routines such as t-tests. It also works well in concert with PowerPoint to produce publication-quality graphics. Simca is a reasonably user-friendly package for more advanced needs, e.g., principle-component analyses, although it's not as versatile for final graphics. Both of these are on a workstation adjacent to - and networked with - Spectrum Mill and Scaffold.

Online Resources:

Multiple Charge States

Multiply-charged ions, e.g., intact proteins, larger peptides, and DNA or RNA oligomers, can usually be deconvoluted (i.e., recalculated to the m/z appropriate for a singly-charged ion) by the workstation software on the mass spectrometers: Analyst for the ABSciex instruments and Mass Hunter for the Agilent instruments. In some cases, especially very noisy data, low-abundances, or overlapping charge envelopes, the spectra need to be deconvoluted manually. We have an interactive spreadsheet routine that can help with this; you can download it here.