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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Comput Biol Chem. Author manuscript; available in PMC Jun 1, 2009.
Published in final edited form as:
PMCID: PMC2487672
NIHMSID: NIHMS53066

MASIC: a software program for fast quantitation and flexible visualization of chromatographic profiles from detected LC-MS(/MS) features

Abstract

Quantitative analysis of liquid chromatography (LC)- mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data is essential to many proteomics studies. We have developed MASIC to accurately measure peptide abundances and LC elution times in LC-MS/MS analyses. This software program uses an efficient processing algorithm to quickly generate mass specific selected ion chromatograms from a dataset and provides an interactive browser that allows users to examine individual chromatograms with a variety of options.

Keywords: Liquid chromatography-tandem mass spectrometry (LC-MS/MS), Quantitation, Proteomics, Quantification, Selected ion chromatogram

1. Introduction

Proteomic studies frequently depend on quantitative information to gain useful biological insights. Several software packages, including ASAPRatio (Li et al., 2003) and RelEx (MacCoss et al., 2003), have been developed to generate quantitative information, generally in the context of relative abundances of isotope-labeled species from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. This approach is limited by the resolution of the mass spectrometer; therefore analysis of isotopic pairs is best accomplished by using high-mass-measurement-accuracy MS (Qian et al., 2005b). Isotopic pairs processing is efficiently managed in-house by an advanced software package called VIPER (Monroe et al., 2007) that is uniquely tailored to the advantages of high resolution and high mass accuracy LC-MS. For characterizing species detected in low-resolution MS/MS analyses, we created the software package MASIC (MS/MS Automated Selected Ion Chromatogram Generator) to enable “label-free” quantitation.

2. Materials and Methods

MASIC evaluates peptide abundances and elution times in an LC-MS/MS analysis and provides the user with a flexible interface for viewing this information. The program first analyzes LC-MS/MS data to generate descriptions for each MS and MS/MS spectrum in terms of scan acquisition time, scan type, total ion intensity, base peak intensity, etc. This information is then used to generate “selected ion chromatograms” (SICs) for each species chosen for MS/MS fragmentation. Chromatographic data such as peak width, height, area, and apex elution time are computed for each selected ion chromatogram. The peak apex is a more accurate measure of elution time than the scan numbers associated with fragmentation spectra, which allows for more precise alignment to LC-MS features in the AMT tag approach (Zimmer et al., 2006). MASIC also generates text files that summarize SIC and MS(/MS) scan information, allowing the user to generate “total ion chromatograms” (TIC) or "base peak intensity" (BPI) chromatogram plots using either a standard graphing program or the PNNL developed software package called Decon2LS (available free of charge at http://ncrr.pnl.gov/software/). The data generated are also written to an .Xml file that can be read by an accompanying executable called MASIC Browser. Both MASIC and MASIC Browser include graphical user interfaces (GUIs) along with Visual Basic .NET DLLs that allow for command line or code-based automation. The compiled executables and the source code are available for download free of charge at http://ncrr.pnl.gov/software/.

2.1 Data Processing

MASIC recognizes .Raw files from Thermo Fisher mass spectrometers, mzXML files (http://sashimi.sourceforge.net/software_glossolalia.html), mzData files (http://www.psidev.info/), and pairs of CDF and Mascot generic format (MGF) files that can be generated for Agilent ion trap mass spectrometers. Each of these formats contains similar information about the raw data, which allows MASIC to determine base peak intensity and m/z, total ion intensity, scan (LC elution) time, scan type, and in the case of MS/MS spectra, parent ion m/z.

MASIC includes a basic compression algorithm to deal with high-resolution data. If more than two consecutive points have zero intensity, the entire range is specified simply by the starting and ending m/z. The program also groups all points within an m/z range specified by the user; example bin sizes are 0.02 m/z for MS spectra and 0.2 m/z for MS/MS spectra. These steps reduce the amount of data used without a significant decrease in data quality, which improves processing rates, eases memory requirements, and reduces output file sizes.

After calculating a noise threshold for each MS parent ion spectrum, MASIC creates the SICs. First, a list of parent ions—m/z values chosen for fragmentation—is compiled, which may be supplemented by custom values provided by the user. These values are grouped into small m/z ranges (e.g., 0.05 m/z), and one SIC is created for each range by searching the MS1 spectra for data within a user-defined tolerance of the median m/z value within the range. In each mass spectrum, the sum of the intensity values for the data within the m/z search window defines that spectrum’s contribution to the SIC. If a spectrum has no data in the m/z window, then the minimum positive intensity across the entire spectrum is taken as its contribution.

Following SIC generation, MASIC analyzes each SIC, which represents a unique LC elution time interval and m/z range, for useful information. First, the software finds the largest peak (that includes the scan number of the original m/z) in the SIC and applies Butterworth data smoothing, as implemented by the XPRESS software package (Han et al., 2001). The peak’s signal-to-noise ratio, full-width-at-half-maximum, and area are calculated using the unsmoothed data, while the smoothed data are used to compute several statistical measures of peak shape. These measures include the Kolmogorov-Smirnov statistic (K-S stat) to estimate peak skew and the computation of the statistical moments, which provide peak area (zeroth moment), center of mass (1st central moment), peak standard deviation (2nd moment), and peak skew (derived from the 3rd moment).

MASIC also searches for spectra with similar parent ion m/z values and elution times. The fragmentation patterns of potentially similar spectra are compared using a Pearson correlation. If the correlation value exceeds a threshold, then the relationship between the spectra is recorded and the peak apex elution time of the spectrum with the largest peak area is used to define the elution time of the other related spectra.

A 3.2 GHz Pentium 4 computer was used to benchmark the performance of MASIC when processing Thermo Fisher .Raw files generated by LCQ, LTQ, and LTQ-FT analyses of a standard peptide mixture (a tryptic digest of a mixture of 12 proteins modeled after the mixture suggested by Purvine et al., 2004) . As shown in Table 1, for separations with ~104 spectra, the time spent loading and analyzing the file was usually less than 10 minutes, and often less than 5 minutes.

Table 1
Processing benchmarks for MASIC using a 3.2 GHz Pentium 4 computer

2.2 MASIC Browser

After the data file has been analyzed and chromatograms created, the MASIC Browser allows the user to examine the chromatograms individually, as shown in Figure 1 and Figure 2. The list of SICs can be sorted by various criteria, including m/z, peak intensity, peak width, peak area, and signal-to-noise ratio. The SICs may also be filtered by intensity, signal-to-noise ratio, or m/z. The Butterworth-smoothed data are shown along with the SIC data by default; however, these data can be either removed from the display or replaced by data processed by Savitzky-Golay smoothing. The user can navigate the display window by zooming and panning to view adjacent regions of the chromatogram. Two floating cursors are provided to help the user visually evaluate the SIC. The initial cursor positions indicate the scan number at the peak apex of the SIC and the scan number of the MS/MS fragmentation event that provided the m/z value used to generate the SIC. The Browser also allows the user to annotate the plot with arrows and text.

Figure 1
MASIC Browser interface. The user can browse the list of selected ion chromatograms, view the statistics for each, and filter the displayed data. As each chromatogram is selected, it is plotted in a separate window (see Figure 2).
Figure 2
Selected Ion Chromatogram plot in MASIC Browser. Both the raw data (line with points) and the smoothed data (no points) are plotted. The user can navigate the chromatogram, zooming, panning, and annotating as desired.

3. Summary

MASIC quickly analyzes raw data from LC-tandem mass spectrometry studies and generates dataset-wide chromatograms and selected ion chromatograms for MS parent ions chosen for MS/MS fragmentation and/or for user-defined m/z values. The results can be visualized using MASIC Browser and are summarized numerically in text files. The abundance and elution time information computed for individual MS features enables more accurate generation of reference databases of mass and (elution) time tags as utilized by the AMT tag approach. The abundance values can also be used to compare peptides between two samples to compute peptide peak area ratios, as demonstrated in a proteomic study analyzing human plasma (Qian et al., 2005a).

Acknowledgements

Portions of this research were supported by the U. S. Department of Energy Office of Biological and Environmental Research Genomes:GtL Program, the NIH National Center for Research Resources (Grant RR018522), and the National Institute of Allergy and Infectious Diseases (NIH/DHHS through interagency agreement Y1-AI-4894-01).

Footnotes

This manuscript has been authored by Battelle Memorial Institute, Pacific Northwest Division, under Contract No. DE-AC05-76RL0 1830 with the U.S. Department of Energy. By accepting the article for publication, the publisher acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes.

Availability: MASIC may be downloaded free of charge at http://ncrr.pnl.gov/software/

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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