Abstract
The complexity of the human proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at >or=1% level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.
Highlights
The complexity of the human proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution
Further complexity is added at several levels mainly in the form of alternative splicing and post-translational modifications (PTMs)
When combining the complexity generated by alternative splicing with that produced by PTMs, the current estimate of the number of different protein molecules expressed in a given individual organism is close to a million, which is roughly 50 forms per gene [6]
Summary
Sample Preparation—500 g of A431 human epidermoid carcinoma and SD1 human acute lymphoblastic leukemia cell lysates was loaded onto four independently prepared one-dimensional SDSPAGE gels (ϳ30 –200 kDa). Nanoflow LC/MS/MS—All experiments were performed on a 7-tesla hybrid linear ion trap Fourier transform mass spectrometer (LTQ FT, Thermo Electron, Bremen, Germany) modified with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). MS analysis was performed using unattended data-dependent acquisition mode in which the mass spectrometer automatically switches between a high resolution survey scan (resolution ϭ 100,000; m/z range, 200 –1600) followed by lower resolution fragmentation spectra (electron capture dissociation [27, 28] followed by collision-activated dissociation; resolution ϭ 25,000) of the two most abundant peptides eluting at a given time. Searches were performed with trypsin specificity [30], and mass tolerance for monoisotopic peptide identification was set to 5 ppm and Ϯ0.02 Da for fragment ions. Parsing of data and statistical analysis of the search results reported by Mascot were performed using the open-source software MSQuant [50]
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