Proteome coverage

The complementary nature of MALDI-MS and ESI-MS-MS can also be exploited to increase the proteome coverage via PSA [36]. A fraction (20%) of the column effluent of a nano-LC column was sent to ESI-MS-MS, while the rest was fractionated onto MALDI spots for automated off-line LC-MALDI-MS and MS-MS analysis. About 63% overlap in protein identified from a digest of mammalian mitochondrial ribosomes was observed, but unique proteins were also identified by either technique. 

Lee et al. [88] discussed optimization of RPLC for quantitative proteomics. They especially paid attention to the fact that both a good chromatographic peak shape must be achieved to enable reliable quantification and the peak capacity must be maximized to enable identification with sufficient proteome coverage. Gradient range and slope were evaluated. 

A.W. Guzzetta, A.S. Chien, A double-vented tetraphasic continuous column approach to MudPIT analysis on long capillary columns demonstrates superior proteomic coverage, J. Proteome Res., 4 (2005) 2412. 

L. Pasa-Tolic R. Harkewicz, G.A. Anderson, M. Tolic, Y. Shen, R. Zhao, B. Thrall, C. Masselon, R.D. Smith, Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FT-ICRMS, J. Am. Soc. Mass Spectrom., 13 (2002) 954. 

In this chapter, we focus on the strategies that have been developed during the past few years to increase the proteome coverage in shotgun proteomics studies (Figure 1). The analytical techniques that might increase the fragmentation rate are outlined briefly in Section 2 (for a comprehensive review,... 

No current analytical strategy is capable of fully resolving complex biological samples. For this reason, orthogonal separation techniques are often combined to maximize peptide separation before the mass spectrometric analysis. The aim is to reduce as much as possible the number of coeluting peptides introduced into the MS at any given time, so as to maximize peptide identification and proteome coverage. 

Other proteolytic enzymes commonly used in proteomics analysis include Lys-C, Lys-N, and chymotrypsin. In general, these enzymes differ by their specificity for cleaving the amide bond before or after a specific residue. For complex protein samples, a combination of highly selective proteases has proved to increase proteome coverage by creating complementary peptides (13). 

Several DDA add-ons and alternatives have been proposed in order to tackle the issues of poor reproducibility and proteome coverage. These studies can be grouped into two categories (i) hypothesis-driven acquisition (HDA), also called targeted acquisition and (ii) data-independent acquisition (DIA). 

Areas of intense research are to increase proteome coverage (especially at low copy numbers per cell) while increasing throughput, and to do so in a quantitative manner. Yates and co-workers have developed a large-scale analytical scheme that combines multidimensional nanoscale LC, MS/MS, and automated sequence analysis referred to as multidimensional protein identification... 

Although mass spectrometry has made significant strides in sensitivity and proteome coverage, a recent report by Weissman and co-workers indicates that significant challenges remain [72]. With the use of a complete fusion, tandem-affinity-purification (TAP)-tag library for the genome of Saccharomyces cere-visiae, protein expression levels were quantitatively determined with Western blots by chemiluminescent detection. These results were compared to MS-based (MudPIT) results [54,73], and the MS data were strongly biased toward the detection of abundant proteins. For the 75% of the proteome that is represented by proteins present at fewer than 5000 copies per cell, only 8% were... 

Increasing Proteomic Coverage by Cellular, Sub-Cellular and Protein Fractionation... 

An alternative approach to increasing proteomic coverage is based on the analysis of isolated intact mitochondrial protein complexes. One strategy is based on the use of sucrose density gradients to separate intact mitochondrial complexes solubilised with n-dodecyl- 3-D-maltoside (Hanson et al., 2001). Initially the proteins from the individual fractions were analysed by 2-DE. However, subsequently this approach has been coupled with 1-D SDS-PAGE and MALDI PMF analysis of tryptic digests of excised protein bands (Taylor et al., 2003). When applied to human heart mitochondria, this... 

Umar, A. Luider, T.M. Foekens, J.A. Pasa-Tolic, L. Nano-LC FT-ICR MS improves proteome coverage attainable for similar to 3000 laser-microdissected breast carcinoma cells. Proteomics 2007, 7(2), 323-329. 

Bodnar WM, Blackburn RK, Krise JM, Moseley MA. Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage. J Am Soc Mass Spectrom. 2003 14(9) 971-9. 

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