Bottom-up proteomics

Millea, K.M., Krull, I.S., Cohen, S.A., Gebler, J.C., Berger, S.J. (2006). Integration of multidimensional chromatographic protein separations with a combined top-down and bottom-up proteomic strategy. J. Proteome. Res. 5, 135-146. 

The enzymes used for bottom-up proteomic studies can be classified as those with specific cleavage specificity and those with nonspecific proteolytic activity. 

Carbonylation occurs by the oxidation of some amino acid side chains into ketone or aldehyde derivatives by reactions with compounds of lipid oxidation or by glycoxidation with reducing sugars. These protein-carbonyl compounds are markers of protein oxidation, and recently, several carbonylated proteins and protein oxidation sites in milk (96), meat (97), and fishes (98) have been identified using a classical bottom-up proteomics approach based on 2-DE and MS/MS. Specific labeling of protein carbonyls using fluorescein-5-thiosemicarbazide has been developed and combined with 2-DE and... 

Millea KM, Krull IS, Cohen SA, Gebler JC, Berger SJ (2006) Inte-gradon of multidimensional chromatographic protein separadons with a combined top-down and bottom-up proteomic sdategy. J Proteome Res 5 135—146. 

It should be noted that ETD is a relatively inefficient process for doubly protonated peptide precursors [M + 2H]2+, which are the ions most commonly found in bottom-up proteomics experiments. This situation may be retrieved, however, by using a supplemental low-energy CID method (ETciD) to target the nondissociated electron transfer (ET) product, [M + 2H]2+. CID of the ET product then yields c- and z-type fragment ions. Swaney etal.110 have reported that in a large-scale analysis of doubly charged tryptic peptides, the use of ETciD resulted in a median sequence coverage of 89% compared to 63 and 77% for ETD and CID, respectively. 

In addition to analyst-derived keratins, proteins/peptides can also be introduced, for example, in bottom-up proteomics where the sample is digested by a proteolytic enzyme (most commonly trypsin). 

Generically, sample preparation in bottom-up proteomics involves the solubilization of proteins from the biological source, protein fractionation, subsequent enzymatic digestion of the proteins, and separation of the resulting peptides. Unique enrichment techniques can be applied in many of these preparatory steps (Figure 5). 

Methods incorporating FT-ICR MS/MS have been applied also to bottom-up proteomic analyes. Hakansson et al. [66] applied ESI FT-ICR and IRMPD MS/MS to the analysis of glycoproteins isolated from human cerebrospinal fluid. Brock and co-workers [103] combined MALDI FT-ICR with SORI-CID. The throughput of this approach is hampered by the timescales associated with SORI-CID. Laskin and co-workers [104] compared approaches utilizing SORI-CID and SID coupled to ESI. The protein identification scores were comparable for the two techniques. SID has the advantage that no pump-down delay is needed and, therefore, more cycles of MS/ MS can be completed. 

In Chapter 4 is discussed the bottom-up or shotgun tand mass spectrometric approach to protein identification and characterization, which is the complementary method to top-down proteomics that is discussed in Chapter 3. In order to overcome the limitations of bottom-up proteomics, chemical derivatization strategies are... 

Bottom-up proteomics. This strategy relies on peptide-level information, such as mass or sequence, to identify a protein. Cnrrently, this strategy dominates proteomics research. In this approach, the protein is digested, typically with trypsin, and the digest is analyzed with an appropriate mass spectrometry system to obtain the peptide masses and sequences. The database search with mass spectrometry data characterizes the protein. More details of the mass spectrometry platforms used in this approach are provided in the following sections. 

How do bottom-up proteomics differ from top-down proteomics ... 

Brock, A., Horn, D.M., Peters, E.C., Shaw, C.M., Ericson, C., Phung, Q.T., and Saloma, A.R. (2003) An automated matrix-assisted laser desorption/ ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometer for bottom-up proteomics. Anal. Chem., 75, 3419-3428. 

Han B, Hare M, Wickramasekara S, Fang Y, Maier CS (2012) A comparative bottom up proteomics strategy for the site-specific identification and quantification of protein modifications by electrophilic lipids. J Proteomics 75(18) 5724-5733.doi 10.1016/j.jprot.2012.07.029... 

Top-down proteomics [1] is the identification and characterization of a mature, intact protein (or proteins) using primarily mass spectrometry (MS)-based techniques and the sequence information contained in genomic/proteomic databases. Unhke bottom-up proteomics [2-4], where a protein (or proteins) is digested into peptides prior to MS or tandem mass spectrometry (MS/MS) analysis [3,4], the top-down approach involves measuring the molecular weight (MW) of the intact, mature protein with its associated posttranslational modifications (PTMs) if any. The intact protein ion is then fragmented in the gas phase in order to determine its amino acid sequence as well as the location and identification of PTMs. From its earliest development, top-down proteomics has been primarily the domain of electrospray ionization (ESI) [5] (which generates mul-... 

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