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Chromatographic alignment improves differential MS CRAWDAD software facilitates label-free quantitation of proteomics mixtures.
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Intensity (×10 6)
ifferential MS (dMS) can detect for both the noninduced and induced peak to get results. MacCoss says, “It’s quantitative differences between samples were run on a reversed-phase very appealing because you’re not relysamples without the need to label the µLC column coupled to a linear ion ing on your ability to detect something analytes. However, variations in the trap quadrupole (LTQ) Orbitrap mass first. You’re just using the statistics to LC retention times can complicate spectrometer. say what’s different.” Data warping this method of label-free quantitation. CR AWDAD identified 5920 statisti- turns out to be particularly useful for To expand the applicability of dMS, cally significant difference regions and low-resolution data because it does not Michael MacCoss and his coldepend on identifying the isotope Aligned (c) Difference region distributions. The software should (b) (a) Unaligned leagues at the University of Washington and the University not be limited by the scanning 10 of Colorado Denver have develspeed of the mass analyzer, and 8 oped chromatogram retentionit is compatible with most mass time alignment and warping spectrometers regardless of reso6 for differential analysis of data lution. Edward Marcotte of the 4 (CR AWDAD), a new suite University of Texas Austin says, “I of software tools that aligns think that what they’re getting is 2 the LC/MS runs and then an increase in peak detection.” He performs label-free quantitasays the method could highlight 57.4 57.7 58.0 57.4 57.7 58.0 57.4 57.7 58.0 tion of peptide mixtures with modified peptides that are diffiRetention time (min) Retention time (min) Retention time (min) dMS (Anal. Chem. 2007, DOI cult to detect by other means. 10.1021/ac701649e). Extracted ion chromatograms for the 2+ ion of the β-galactosiCR AWDAD cannot quantiMany researchers perform dase peptide LWSAEIPNLYR at m/z 681.3–681.4. (a) Unaligned tate relative differences in peptide feature-based alignments, which and (b) aligned replicates of an induced sample. Replicates abundance if the peptides elute in apply a regression to match are black; the mean is red. (c) Aligned replicates from induced a different order or are resolved peaks or isotope clusters from (red) and noninduced (blue) samples. The difference region is in one run and unresolved in the MS scan or peptide identishaded in red. another. “We can’t improve the ties from the MS/MS scan. resolution by warping,” MacCoss Instead, MacCoss and colleagues chose detected 95 proteins in which two or says. “It can’t actually make bad chroa signal-based alignment that breaks up more peptides changed in abundance. matography good.” Any errors or losses the LC/MS run into bins of time and The alignment resulted in a 4-fold dethat occur during the sample preparam/z and then optimizes the similarcrease in mean retention time RSD, a tion will be detected as differences in ity scores between two runs through a 5-fold increase in S/N, and a 6-fold inthe overall analysis, so technical as well dynamic time-warping algorithm. “Dycrease in the number of statistically sigas biological replicates must be ananamic time warping is actually a pretty nificant differences detected before and lyzed. Jaffe would like to see how the good technique for things that don’t after the lac operon was activated. software handles noise contributed by behave well, like chromatography,” says The team was surprised that MS/MS biological replicates and what it can do Jacob Jaffe of the Broad Institute. After spectra were automatically acquired for with more complex mixtures, such as aligning LC/MS scans to a master scan, a mere 38% of the difference regions human serum. CR AWDAD identifies statistically sigand that only 23% of the difference reTo decrease the processing time, the nificant regions of difference and checks gions could be assigned peptide identiresearchers plan to change the CR AWwhether the instrument acquired an fications based on the MS/MS spectra. DAD program language from Python MS/MS spectrum that can help identify “If 75% of all the things that are chang- to C++. Now, the team is testing for the peptide. ing between any two sets of samples robustness and user-friendliness of the The software was tested by analyzing cannot be assigned an identity based on method. By making CR AWDAD freely tryptic peptides from a well-characterthe MS/MS spectra, by relying on data- available on the web for nonprofit use, ized example of gene regulation in dependent acquisition to find things, they hope to see it implemented more bacteria. The group detected differences we’re missing a lot,” observes MacCoss. widely to address interesting biological in proteins before and after induction One of the principal advantages of this questions. a of the lac operon. Technical replicates technique is that you don’t have to see a —Christine Piggee © 2008 American Chemical Societ y
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