Update 06.proteolysis.md

content/06.proteolysis.md

@@ -10,25 +10,25 @@ This section summarizes potential choices and their strengths and weaknesses.
 
 Trypsin is the most common choice of protease for proteome hydrolysis [@DOI:10.1002/mas.21376].
 Trypsin is favorable because of its specificity, availability, efficiency and low cost. 
-Trypsin cleaves at the C-terminus of basic amino acids, Arg and Lys. 
+Trypsin cleaves at the C-terminus of basic amino acids, Arg and Lys, if not immediately followed by proline. 
 Many of the peptides generated from trypsin are short in length (less than ~ 20 amino acids), which is ideal for chromatographic separation, MS-based peptide fragmentation and identification by database search.
 The main drawback of trypsin is that majority (56%) of the tryptic peptides are ≤ 6 amino acids, and hence using trypsin alone limits the observable proteome [@PMID:20113005; @PMID:25823410; @PMID:30687733].
 This limits the number of identifiable protein isoforms and post-translational modifications.
 
 Although trypsin is the most common protease used for proteomics, in theory it can only cover a fraction of the proteome predicted from the genome [@DOI:10.1155/2014/960902]. 
 This is due to production of peptides that are too short to be unique, for example due to R and K immediately next to each other. 
-Peptides below a certain length are likely to happen many times in the whole proteome, meaning that even if we identify them we cannot know their origin protein. 
-In protein regions devoid of R/K, trypsin may also result in very long peptides that are then lost do to irreversible binding to the solid phase extraction device, or that become difficult to identify due to complicated fragmentation patterns. 
+Peptides below a certain length are likely to occur many times in the whole proteome, meaning that even if we identify them we cannot know their protein of origin. 
+In protein regions devoid of R/K, trypsin may also result in very long peptides that are then lost due to irreversible binding to the solid phase extraction device, or that become difficult to identify due to complicated fragmentation patterns. 
 Thus, parts of the true proteome sequences that are present are lost after trypsin digestion due to both production of very long and very short peptides. 
 
-Many alternative proteases are available with different specificities that complement trypsin to reveal different proteomic sequences [@PMID:12643544; @PMID:20113005], which can help distinguish protein isoforms [@PMID:27123950] (Figure 2).
+Many alternative proteases are available with different specificities that complement trypsin to reveal different protein sequences [@PMID:12643544; @PMID:20113005], which can help distinguish protein isoforms [@PMID:27123950] (Figure 2).
 The enzyme choice mostly depends on the application.
-In general, for a mere protein identification mostly trypsin is the choice due to the reasons aforementioned.
+In general, for a mere protein identification, trypsin is often chosen due to the aforementioned reasons.
 However, alternative enzymes can facilitate _de novo_ assembly when the genomic data information is limited in the public database repositories [@pmid:31615963; @pmid:30622160; @pmid:29990557; @doi:10.1016/j.actatropica.2022.106324; @DOI:10.1021/pr400173d].
 Use of multiple proteases for proteome digestion also can improve the sensitivity and accuracy of protein quantification [@PMID:30336047].
 Moreover, by providing an increased peptide diversity, the use of multiple proteases can expand sequence coverage and increase the probability of finding peptides which are unique to single proteins [@DOI:10.1021/acs.jproteome.9b00330; @DOI:10.1074/mcp.M113.034710; @DOI:10.1155/2014/960902]. 
 A multi-protease approach can also improve the identification of N-Termini and signal peptides for small proteins [@DOI:10.1021/acs.jproteome.1c00115]. 
-Overall, integrating multiple-protease data can increase the number of proteins identified [@DOI:10.3390/ijms20225630; @DOI:10.1074/mcp.M113.035170], the number of identified post-translational modifications detected [@DOI:10.1021/acs.jproteome.9b00330; @DOI:10.1016/j.celrep.2015.05.029; @DOI:10.1074/mcp.M113.034710] and decrease the ambiguity of the protein group list [@DOI:10.1021/acs.jproteome.9b00330].
+Overall, integrating multiple-protease data can increase the number of proteins identified [@DOI:10.3390/ijms20225630; @DOI:10.1074/mcp.M113.035170], increase the identified post-translational modifications [@DOI:10.1021/acs.jproteome.9b00330; @DOI:10.1016/j.celrep.2015.05.029; @DOI:10.1074/mcp.M113.034710] and decrease the ambiguity of the inferred protein groups [@DOI:10.1021/acs.jproteome.9b00330].
 
 ![**Multiple protease proteolysis improves protein inference**
 The use of other proteases beyond Trypsin such as Lysyl endopeptidase (Lys-C), Peptidyl-Asp metallopeptidase (Asp-N), Glutamyl peptidase I, (Glu-C), Chymotrypsin, Clostripain (Arg-C) or Peptidyl-Lys metalloendopeptidase (Lys-N) can generate a greater diversity of peptides. 
@@ -42,7 +42,7 @@ A major advantage of Lys-C is its resistance to denaturing agents, including 8 M
 Trypsin is less efficient at cleaving Lys than Arg, which could limit the quality of quantitation from tryptic peptides.
 Hence, to achieve complete protein digestion with minimal missed cleavages, Lys-C is often used simultaneously with trypsin digestion [@PMID:23017020].
 
-Alpha-lytic protease (aLP) is also secreted by the soil bacterial _Lysobacter enzymogenesis_ [@PMID:3053694].
+Alpha-lytic protease (aLP) is another protease secreted by the soil bacterial _Lysobacter enzymogenesis_ [@PMID:3053694].
 Wild-type aLP (WaLP) and an active site mutant of aLP, M190A (MaLP), have been used to expand proteome coverage [@DOI:10.1074/mcp.m113.034710].
 Based on observed peptide sequences from yeast proteome digestion, WaLP showed a specificity for small aliphatic amino acids like alanine, valine, and glycine, but also threonine and serine. 
 MaLP showed specificity for slightly larger amino acids like methionine, phenylalanine, and surprisingly, a preference for leucine over isoleucine. 
@@ -59,7 +59,7 @@ Studies also suggest that Asp-N cleaves at the amino terminus of glutamate when
 Asp-N often leaves many missed cleavages [@PMID:27123950].
 
 Chymotrypsin or chymotrypsinogen A is a serine protease obtained from porcine or bovine pancreas with an optimum pH range from 7.8 to 8.0 [@PMID:3555886]. 
-It cleaves at the C-terminus of hydrphobic amino acids Phe, Trp, Tyr and barely Met and Leu residues.
+It cleaves at the C-terminus of hydrophobic amino acids Phe, Trp, Tyr and barely Met and Leu residues.
 Since the transmembrane region of membrane proteins commonly lacks tryptic cleavage sites, this enzyme works well with membrane proteins having more hydrophobic residues [@PMID:24870543; @PMID:24696503; @PMID:27123950].
 The chymotryptic peptides generated after proteolysis will cover the proteome space orthogonal to that of tryptic peptides both in a quantitative and qualitative manner [@PMID:24290761; @PMID:22669647; @PMID:24696503]
 
@@ -75,8 +75,8 @@ In addition, it can also cleave modified amino acids such as methylated or dimet
 
 Peptidyl-Lys metalloendopeptidase, or Lys-N, is an metalloprotease obtained from _Grifola frondosa_ [@PMID:19195997]. 
 It cleaves N-terminally of Lys and has an optimal activity at pH 9.0.
-Unlike trypsin, Lys-N is more resistant to denaturing agents and can be heated up to 70 °C [@PMID:25823410].
-Reports suggest that the peptides generated after Lys-N digestion produces more of c-type ions in a ETD-based mass spectrometer [@PMID:18425140]. 
+Unlike trypsin, Lys-N is more resistant to denaturing agents and can be heated up to 70°C [@PMID:25823410].
+Peptides generated from Lys-N digestion produce more c-type ions using ETD fragmentation [@PMID:18425140]. 
 Hence this can be used for analysing PTMs, identification of C-terminal peptides and also for _de novo_ sequencing strategies [@PMID:18425140; @PMID:20953479].
 
 Pepsin A, commonly known as pepsin, is an aspartic protease obtained from bovine or porcine pancreas [@PMID:12089768].
@@ -91,6 +91,6 @@ It is a member of the subtilisin family of proteases and is relatively unspecifi
 The optimal enzyme activity is between pH 7.5 and 12.
 Proteinase K is used at low concentrations for limited proteolysis (LiP) and the detection of protein structural changes in the eponymous technique LiP-MS [@PMID:29072706]. 
 
-Although different specificity is useful in theory to enable improved proteome sequence coverage, there are practical challenges because most of the standard workflow is optimized for tryptic peptides. 
+Although different specificity is useful in theory to enable improved proteome sequence coverage, there are practical challenges because most standard workflows are optimized for tryptic peptides. 
 For example, peptides that lack a c-terminal positive charge due to arginine or lysine side chains can have a less pronounced y-ion series.
 This can lead to lower scoring peptide-spectra matches because some peptide identification algorithms preferentially score y ions higher.