Roles of the Carboxypeptidase Family in Protein C-Terminal Processing and Experimental Analysis
Roles of the Carboxypeptidase Family in Protein C-Terminal Processing and Experimental Analysis
Carboxypeptidases are a class of exopeptidases that act on amino acid residues at the C-terminus of peptides or proteins, and they have important value in protein processing, bioactive peptide maturation, terminal structure trimming, and experimental analysis. Unlike endoproteases, which generate peptide boundaries through internal cleavage, carboxypeptidases are more closely associated with precise removal of terminal residues and control of C-terminal consistency. In vivo, carboxypeptidases participate in precursor protein maturation, regulation of inflammatory mediators, and protein degradation; in experimental systems, they are commonly used for C-terminal structure confirmation, control of terminal heterogeneity, peptide end-trimming, and multi-enzyme sequential processing.
Keywords: carboxypeptidase family; metallocarboxypeptidase; serine carboxypeptidase; C-terminal processing; protein modification; multi-enzyme combination; mass spectrometry; terminal heterogeneity
I. Classification and Functional Features of the Carboxypeptidase Family
1.1 Major Types Based on Catalytic Mechanism
(1) Metallocarboxypeptidases
Metallocarboxypeptidases are characterized by the participation of metal ions such as Zn2+ in catalysis. Common members include pancreatic carboxypeptidase A, carboxypeptidase B, and certain regulatory carboxypeptidases. These enzymes generally display good activity under neutral to mildly alkaline conditions and are relatively sensitive to metal chelators. In experimental techniques, metallocarboxypeptidases are often used as terminal-trimming tools for studying the composition and changes of protein or peptide C-termini.
(2) Serine carboxypeptidases
Serine carboxypeptidases use a serine residue as the catalytic core, and their enzymatic mechanism differs substantially from that of metallocarboxypeptidases. Compared with metallocarboxypeptidases, serine carboxypeptidases are often more active under mildly acidic conditions and are suitable for certain specialized buffer systems or acidic processing environments. However, they generally require tighter methodological control with respect to substrate conformation sensitivity and reaction parameters.
1.2 Functional Classification Based on Biological Role
(1) Carboxypeptidases related to digestive degradation
Represented by carboxypeptidase A and carboxypeptidase B, these enzymes participate in terminal degradation of dietary proteins in vivo and can be repurposed as experimental processing tools in vitro. Among them, carboxypeptidase B is particularly practical for controlling residual C-terminal Lys or Arg because of its strong preference for basic residues.
(2) Carboxypeptidases related to maturation processing
Some carboxypeptidases participate in the maturation of precursor proteins and precursor peptides, often acting in concert with endoproteases. After internal cleavage, they remove Lys or Arg residues remaining at the C-terminus, thereby generating active products closer to the natural mature state.
(3) Carboxypeptidases related to homeostatic regulation
Certain plasma or membrane-associated carboxypeptidases participate in terminal trimming of inflammatory mediators, complement components, or the kinin system. Their role is more closely related to fine control of the duration and intensity of signaling molecule activity. The research value of these carboxypeptidases lies primarily in regulatory mechanisms and physiological functions.
II. Substrate Preference and Experimental Boundaries
2.1 C-Terminal Residue Preferences of Different Types of Carboxypeptidases
(1) Carboxypeptidase A–type enzymes
Carboxypeptidase A–type enzymes preferentially remove hydrophobic or aromatic amino acid residues from the C-terminus, such as Phe, Tyr, Trp, Leu, and Ile. They are therefore suitable for analysis of hydrophobic C-terminal residues, stepwise C-terminal sequencing, and certain terminal-trimming applications.
(2) Carboxypeptidase B–type enzymes
Carboxypeptidase B–type enzymes preferentially act on the basic C-terminal residues Lys and Arg. Experimentally, they are commonly used to remove charge heterogeneity caused by terminal basic residues, verify the presence of terminal Lys or Arg, and simulate tail-removal steps in certain maturation processes. Because this substrate specificity is relatively clear, these enzymes are highly practical in protein analysis, sample consistency treatment, and pre–mass spectrometry workflows.
(3) Serine carboxypeptidases
Serine carboxypeptidases generally have a broader substrate range, but their actual reaction performance is more strongly influenced by system pH, substrate conformation, and buffer environment. Under specific acidic conditions, they may be used as terminal-processing tools, but experimental design usually requires more refined parameter optimization.
2.2 Cleavability Does Not Necessarily Mean Experimental Controllability
(1) Accessibility of the C-terminus affects cleavage efficiency
For naturally folded proteins, protein complexes, or highly glycosylated substrates, C-terminal residues may be sterically shielded, thereby reducing carboxypeptidase cleavage efficiency. Accordingly, low cleavage efficiency does not necessarily mean that the target terminal residue is absent; it may instead reflect a problem of structural accessibility.
(2) Adjacent sequence and local conformation exert major effects
The charge, steric bulk, flexibility, and secondary structure of the upstream C-terminal sequence may all affect substrate binding and catalytic efficiency. Even when terminal residues are the same, different proteins or peptides may still exhibit substantially different cleavage rates.
(3) Consecutive cleavable residues may lead to over-trimming
If multiple target residues susceptible to cleavage occur consecutively at the C-terminus, such as ...-Arg-Lys, ...-Lys-Lys, or consecutive hydrophobic residues, carboxypeptidases may perform sequential removal. In such cases, control of the experimental endpoint is often more important than merely reducing enzyme amount. This is especially true when processing terminal Lys/Arg, where careful definition of the reaction time window and intermediate monitoring are essential.
2.3 Type–Preference–Application Comparison Table
Category | Preferred C-terminal residues | Typical technical applications | Main methodological concerns |
Metallocarboxypeptidase A-type | Mainly hydrophobic and aromatic residues | Stepwise C-terminal degradation sequencing; terminal trimming; conformational accessibility probing | Metal dependence; substrate accessibility; avoidance of over-degradation through careful timing |
Metallocarboxypeptidase B-type | Lys, Arg | Removal of terminal Lys/Arg; control of terminal heterogeneity; C-terminal confirmation | Over-trimming due to consecutive cleavable residues; combined charge-profile and MS-based QC |
Maturation-processing carboxypeptidases | Mostly Lys/Arg tails remaining after endoproteolytic processing | Simulation of maturation pathways; mature peptide preparation; precursor processing studies | Order of sequential processing with endoproteases; consistency of endpoint and orthogonal functional validation |
Serine carboxypeptidases | Broader range, but highly sensitive to system conditions | Terminal processing under acidic conditions; coupling with chemical modification workflows | pH window and buffer system; substrate conformation sensitivity; side reactions and specificity boundaries |
III. Technical Position of Carboxypeptidases in Protein Processing
3.1 Functional Complementarity with Endoproteases
(1) Division of labor between boundary generation and terminal trimming
Endoproteases are mainly responsible for generating peptide boundaries at specific internal sites, whereas carboxypeptidases further trim the newly formed C-termini. Thus, in protein processing and experimental analysis, they are generally not interchangeable, but instead serve complementary roles in sequential workflows.
(2) Improving consistency of processing endpoints
In multi-step processing workflows, internal cleavage may leave undesired terminal residues, thereby increasing product heterogeneity. Introducing carboxypeptidase treatment at this stage can reduce differences in terminal composition and improve sample consistency. For samples containing terminal Lys or Arg heterogeneity, carboxypeptidase B–type enzymes are particularly suitable for post-cleavage refinement.
3.2 Sequential Relationship with Precursor Protein Maturation
(1) Tail removal after endoproteolytic cleavage is a common maturation logic
Many precursor proteins transiently retain Lys or Arg residues at newly formed C-termini after internal cleavage, and these residues are subsequently removed by carboxypeptidases to generate the mature active product. This logic is found not only in physiological systems, but can also be simulated in in vitro processing studies.
(2) Reconstitution of maturation workflows in vitro
When studying precursor protein maturation mechanisms or preparing mature peptide products, one may first use an endoprotease to generate boundaries and then employ a carboxypeptidase selective for Lys or Arg to remove terminal tails. This strategy helps generate target mature forms more accurately and can be combined with mass spectrometry or bioactivity assays to verify whether the processing endpoint is as expected.
IV. Common Uses of Carboxypeptidases in Experimental Analysis
4.1 Activity Assays and Kinetic Characterization
(1) Colorimetric or fluorometric assays using synthetic substrates
Colorimetric or fluorometric assays based on synthetic substrates are suitable for enzyme activity comparison, condition screening, and inhibitor evaluation. These methods are simple and relatively high-throughput, but they provide limited information about the conformation and accessibility of real protein substrates.
(2) HPLC or LC–MS product analysis
Direct observation of product changes after terminal residue removal by chromatographic or mass spectrometric methods allows more accurate assessment of actual carboxypeptidase processing outcomes. These methods are particularly suitable for studying terminal Lys/Arg removal and analyzing charge heterogeneity.
(3) Time-course analysis for C-terminal confirmation
By setting multiple reaction time points and observing stepwise mass changes of peptides, one can infer the composition of the C-terminal residues. This approach is suitable for C-terminal confirmation and truncation-pattern studies, but it requires strict control of the reaction window to avoid complexity arising from sequential cleavage.
4.2 Optimization of Protein Sample Consistency and Mass Spectrometric Analysis
(1) Controlling heterogeneity caused by terminal basic residues
Some recombinant proteins, antibodies, or protein fragments may retain varying numbers of terminal Lys residues after preparation, thereby generating charge heterogeneity. Mild treatment with carboxypeptidase B–type enzymes can reduce this difference and improve the consistency of downstream ion-exchange analysis, peptide mapping, and mass spectrometric interpretation.
(2) Assisting confirmation of C-terminal processing status
When the research goal is to determine whether a protein C-terminus has been processed, truncated, or retains specific Lys/Arg residues, carboxypeptidases can serve as directed validation tools. If the focus is on basic residues, carboxypeptidase B–type enzymes are usually more specific.
(3) Combined use with other proteases to improve analytical resolution
In analysis of complex substrates, complementary peptide sets may first be generated with endoproteases, after which carboxypeptidases may be introduced for terminal refinement according to the experimental objective. Such combinations can improve sample consistency, but their potential effects on preservation of key site information must also be evaluated.
V. Selection of Carboxypeptidases in Multi-Enzyme Strategies
5.1 Combination with Common Endoproteases
(1) Fragment first, refine later
A common experimental design is to first generate an analyzable peptide set using endoproteases, and then use carboxypeptidases to refine the termini of selected peptides. This strategy is especially practical when both sample complexity control and terminal consistency are required.
(2) Improving the ability to detect conformational changes
Endoproteases reflect the overall distribution of cleavage sites, whereas carboxypeptidases reflect terminal exposure. Their combined use can therefore help analyze substrate conformational changes, shielding effects due to binding, or local accessibility differences.
5.2 Combination with Endoproteases of Lower Cleavage-Site Density
(1) Optimizing peptide length distribution
When endoproteases such as trypsin generate peptides that are too short or have overly dense cleavage patterns, an endoprotease with lower cleavage-site density may be used first to produce longer fragments, followed by limited terminal trimming with carboxypeptidases. This can improve the balance between analytical coverage and spectral interpretability.
(2) Enhancing reproducibility of peptide sets
In quantitative proteomics or structural characterization, a stable and reproducible peptide set is often more valuable than simply increasing the number of cleavage sites. If the target sample contains terminal Lys or Arg heterogeneity, appropriate introduction of carboxypeptidase B–type enzymes can improve consistency across different sample batches.
5.3 Coupling with Sample Pretreatment Steps
(1) Moderately increasing terminal accessibility
For substrates that are tightly folded, highly glycosylated, or strongly associated in complexes, moderate reduction, mild denaturation, or deglycosylation may improve exposure of the C-terminus and thereby increase carboxypeptidase cleavage efficiency.
(2) Balancing accessibility with native relevance
If the research question depends on native conformation, pretreatment strength must be strictly controlled to avoid introducing artificial conformational bias. It is therefore advisable to include untreated controls and use the observed differences to define the effect of pretreatment.
VI. Selection Strategies for Different Experimental Objectives
6.1 Experimental Objectives Focused on Terminal Lys or Arg Processing
(1) Situations suitable for choosing carboxypeptidase B–type enzymes
When the objective is to remove terminal Lys or Arg, control charge heterogeneity, verify the presence of terminal basic residues, or simulate tail-removal steps in maturation processing, carboxypeptidase B–type enzymes are generally the most direct and targeted choice.
(2) Common QC readouts
① whether the charge profile of the sample becomes more convergent;
② whether C-terminal peptides become more uniform;
③ whether MS-observed mass changes match expectation;
④ whether functional readouts remain stable.
6.2 Experimental Objectives Focused on C-Terminal Structure Confirmation
(1) Selecting the enzyme according to residue type
If the goal is to infer hydrophobic or aromatic terminal residues, carboxypeptidase A–type enzymes should generally be preferred. If the focus is on Lys or Arg, carboxypeptidase B–type enzymes are more appropriate. Choosing the enzyme according to residue type improves interpretive clarity.
(2) Control of the reaction endpoint is more important than maximizing reaction strength
C-terminal confirmation experiments emphasize interpretability rather than maximal cleavage. Therefore, priority should be given to time-course design and endpoint control rather than indiscriminately increasing enzyme amount or prolonging reaction time.
6.3 Experimental Objectives Focused on In Vitro Simulation of Maturation Processing
(1) Constructing an ordered sequential workflow
In vitro simulation of precursor protein maturation can follow a “boundary generation by internal cleavage–tail removal by carboxypeptidase” design. When the terminal tail consists mainly of Lys or Arg, introduction of carboxypeptidase B–type enzymes is particularly helpful for obtaining products close to the natural mature form.
(2) Confirming the degree of maturation using multidimensional readouts
Simulation of maturation processing should not rely on a single MS result alone. Product ratios, functional activity, receptor-binding ability, or other biological readouts should also be considered collectively to determine whether processing has been completed.
VII. Experimental Considerations and Risk Control
7.1 Metal Dependence and System Compatibility
(1) Attention to interference from chelators
For metallocarboxypeptidases, chelators such as EDTA and EGTA may markedly affect enzyme activity. Therefore, when designing the system, one should check in advance whether the buffer, sample stock solution, or pretreatment workflow contains such components.
(2) Attention to pH and ionic strength windows
Different carboxypeptidases differ in their tolerance to pH and ionic strength. It is advisable to first perform small-scale condition screening to identify the linear reaction range before proceeding to formal experiments, thereby reducing errors caused by system drift.
7.2 Troubleshooting When Cleavage Efficiency Is Low
(1) First consider structural shielding factors
If carboxypeptidase cleavage efficiency is low, one should first assess whether the substrate C-terminus is shielded by folding, glycosylation, or complex formation. If necessary, mild denaturation or prior endoproteolytic processing may be attempted to improve terminal accessibility.
(2) Check for system inhibition and enzyme activity loss
In addition to structural factors, one should also examine whether the buffer contains inhibitory components and whether the enzyme preparation itself has lost activity due to freeze–thaw cycles, improper storage, or prolonged standing. Verification of enzyme activity with a standard substrate before critical experiments is generally advisable.
7.3 Preventing Over-Trimming
(1) Rely primarily on time control
Most over-trimming problems arise not simply from excessive enzyme amount, but from excessive reaction time. Therefore, it is recommended to determine a suitable endpoint by preliminary time-course experiments and to terminate the reaction promptly during formal experiments.
(2) Extra caution is required for consecutive target residues
When multiple Lys or Arg residues are present consecutively at the C-terminus, carboxypeptidase B–type enzymes are more likely to perform sequential cleavage. Such samples should be monitored more frequently during the reaction, and terminal changes should be followed in real time using MS or chromatographic methods.
VIII. Aladdin-Related Products
8.1 Experimental Carboxypeptidase Enzyme Products
Catalog No. | Product Name | CAS No. | Grade and Purity |
Carboxypeptidase A from bovine pancreas | 11075-17-5 | ActiBioPure™, Bioactive, EnzymoPure™, ≥50 U/mg protein | |
Carboxypeptidase A from bovine pancreas | 11075-17-5 | EnzymoPure™, ≥20 unit/mg protein | |
Carboxypeptidase A from bovine pancreas | 11075-17-5 | (Type II-PMSF treated), ≥20 units/mg protein | |
Carboxypeptidase B | 9025-24-5 | Animal Free, Carrier Free, Bioactive, Recombinant, ActiBioPure™, EnzymoPure™, ≥90%(SDS-PAGE), ≥150 U/mg protein, expressed in Pichia pastoris | |
Carboxypeptidase B | 9025-24-5 | EnzymoPure™, Native, ≥170 units/mg protein, from porcine pancreas | |
Recombinant Carboxypeptidase B | 9025-24-5 | EnzymoPure™, ActiBioPure™, Bioactive, Animal Free, Carrier Free, suitable for mass spectrometry (MS), Recombinant, ≥95%(HPLC), ≥200 U/mg protein, expressed in E.coli | |
Carboxypeptidase B, Porcine Pancreas | 9025-24-5 | ≥100 units/mg protein | |
Recombinant Carboxypeptidase B | 9025-24-5 | EnzymoPure™, ≥170 units/mg protein | |
Carboxypeptidase G | 9074-87-7 | Bioactive, Recombinant, ActiBioPure™, High Performance, EnzymoPure™, ≥90%(SDS-PAGE), ≥10 U/mg enzyme powder; ≥50 U/mg protein, expressed in E.coli | |
Carboxypeptidase Y, Baker's yeast (S. cerevisiae) | — | — |
8.2 C-Terminal Exopeptidase Experimental Toolbox: Substrates, Readouts, and Metal-Dependence Control
Name | CAS No. | Technical Focus | Key Purpose | Usage Notes |
Hippuryl-L-Arginine | Carboxypeptidase substrate (basic terminal pathway) | Common model substrate for establishing linear range, batch activity verification, and kinetic comparison | Use a time gradient to lock linear range; include both “substrate blank + enzyme-free blank” to avoid misinterpreting background hydrolysis as enzyme activity | |
L-Arginine | Carboxypeptidase product standard | Standard for free Arg after terminal removal; used for derivatization/colorimetric quantification and reaction progress monitoring | For samples with high Arg background, perform blank correction; recommended to build standard curve in parallel with Lys | |
L-Lysine | Carboxypeptidase product standard | Standard for free Lys after terminal removal; used for endpoint determination and sample consistency evaluation (charge heterogeneity / residual tail) | Prepare in same matrix (buffer/salt) to reduce slope drift; verify endpoint with time-point check | |
Ninhydrin | Carboxypeptidase readout (free amino acid colorimetry) | Quantifies free amino acids released by exopeptidase; suitable for activity measurement, reaction monitoring, and inter-batch comparison | Include “sample blank + reagent blank”; build separate curves for Lys and Arg to avoid system bias | |
o-Phthalaldehyde (OPA) | Carboxypeptidase readout (amine fluorescence derivatization) | High-sensitivity quantification of free amines/amino acids; improves resolution for low-activity samples and short reaction windows | Fix derivatization time and temperature; for high amine background, reinforce blank correction and verify dilution linearity and recovery | |
Fluorescamine | Carboxypeptidase readout (rapid fluorescence quantification) | Rapid micro-amine quantification; suitable for high-throughput screening of exopeptidase conditions (pH/metal/inhibitor) | Sensitive to pH and buffer composition; use same batch reagents and reaction time; add standards if needed to monitor matrix effect | |
FMOC-Cl | Carboxypeptidase readout (HPLC derivatization) | Accurate quantification of amino acids/small peptides after derivatization; used for strict comparison of “pre- vs post-processing” trimming | Control derivatization completeness and byproducts; include internal standard and perform spike-recovery to assess matrix effects | |
Dansyl chloride | Carboxypeptidase readout (qualitative/quantitative derivatization) | Confirms amine signal changes due to terminal trimming; compatible with LC/fluorescence detection | Maintain consistent conditions (pH/time/temperature); light-sensitive, handle and store protected from light | |
Norvaline | Carboxypeptidase internal standard | Amino acid internal standard; improves quantification accuracy and inter-batch comparability | Select internal standard well-separated from target peaks; addition time fixed (pre-reaction or post-termination) and documented in SOP | |
GEMSA (2-Guanidinoethylmercaptosuccinic acid) | Carboxypeptidase inhibition control (basic terminal pathway) | Establish specific negative control, verify exopeptidase-driven reaction, and assess inhibition conditions | Use both “pre-incubation + simultaneous addition” modes; run alongside metal-chelation control for proper attribution | |
1,10-Phenanthroline | Carboxypeptidase metal dependence validation/termination | Chelation condition for metal-dependent carboxypeptidases; rapid termination or verification of metal dependence | After termination, desalting/purification before MS recommended; assess potential UV/fluorescence interference | |
2,2′-Bipyridine | Carboxypeptidase metal environment variable | Metal chelator variable to dissect metal environment effects on exopeptidase activity/selectivity | Use low concentration gradients with same pH/ionic strength controls; avoid misinterpreting salt effects as metal effects | |
Zinc chloride | Carboxypeptidase metal supplementation/reactivation | Provides Zn²⁺ to verify metal dependence or restore activity after chelation | Prefer low-concentration gradient; confirm absence of strong chelators; run “Zn-supplemented blank” in parallel | |
Zinc sulfate heptahydrate | Carboxypeptidase zinc source control | Alternative Zn²⁺ source for comparing activity recovery and system compatibility | Run in parallel with ZnCl₂ to separate anion effects; monitor solution freshness and contamination (containers/water) | |
Ammonium bicarbonate | Carboxypeptidase–MS compatible buffer | Volatile buffer for direct drying and MS injection; suitable for peptide mapping/proteomics | Maintain consistent concentration and pH throughout; desalting may be needed to reduce residual salts affecting ionization and column life | |
Ammonium acetate | Carboxypeptidase–MS compatible salt | Volatile salt to improve post-reaction LC-MS compatibility; supports direct injection workflow | Record ionic strength and control salt load; avoid high salt in MS; dilute/desalt and run system blanks if necessary | |
Ammonium formate | Carboxypeptidase–acidic LC compatible buffer | Volatile buffer compatible with acidic LC; coordinates with acidic termination/LC workflow | Link with termination strategy; monitor background peaks and ionization efficiency; include method blanks and column equilibration | |
HEPES | Carboxypeptidase reaction buffer (pH stabilization) | Stabilizes pH under neutral to mildly alkaline conditions; reduces drift effects on exopeptidase activity and endpoint determination | Fix buffer strength and temperature; avoid changing buffer batch for inter-batch comparison; note chelator/metal carryover | |
Urea | Carboxypeptidase substrate accessibility modulation | Mild denaturation to expose C-terminal, improving terminal accessibility and trimming efficiency (folded proteins/glycoproteins) | Dilute denatured sample to enzyme-tolerable range before addition; keep untreated control to assess conformation effects | |
Guanidine hydrochloride | Carboxypeptidase hard substrate unfolding control | For highly shielded substrates, enhances C-terminal exposure and exopeptidase accessibility (extreme/ mechanistic control) | Dilute or desalt before enzyme addition; avoid residual reagent affecting enzyme activity or downstream LC-MS compatibility | |
PMSF | Carboxypeptidase background control (serine proteases) | Inhibit serine protease background, reduce endoproteolysis interference, improve attribution of terminal trimming | Not for main metal-dependent exopeptidase termination; include “with/without PMSF” control to exclude endoprotease contamination | |
E-64 | Carboxypeptidase background control (cysteine proteases) | Inhibit cysteine protease contamination; reduce non-target hydrolysis interference | Recommended for complex samples/natural enzyme preps; combine with PMSF/pepstatin for cleaner background | |
Pepstatin A | Carboxypeptidase background control (aspartic proteases) | Prevent additional degradation by aspartic proteases affecting terminal analysis | Used as background control, not main termination; verify solvent/volume compatibility with LC-MS | |
Aprotinin | Carboxypeptidase background control (trypsin-like) | Inhibit trypsin-like/chymotrypsin-like activity during reaction window | Suitable for sample prep; include “no inhibitor” control to assess downstream digestion/readout effects | |
Trypsin | Carboxypeptidase sequential processing (endo→exo) | Generate peptide boundaries with endopeptidase, then C-terminal refinement for improved peptide map consistency and reduced charge heterogeneity | Maintain “endo first, exo second” order; exopeptidase treatment recommended as short gradient with monitoring to avoid over-trimming consecutive residues |
The carboxypeptidase family has clear and distinctive technical value in protein C-terminal processing systems. Compared with endoproteases, which define peptide boundaries through internal cleavage, carboxypeptidases are more suitable for terminal trimming, control of terminal consistency, and confirmation of C-terminal structure. Different types of carboxypeptidases each possess characteristic catalytic mechanisms, substrate preferences, and experimental requirements. Among them, carboxypeptidases that preferentially remove Lys and Arg are particularly representative in processing terminal basic residues, controlling sample heterogeneity, and simulating maturation processing.
For more related articles, please see below:
[1] Why Perform N-Terminal Acetylation and C-Terminal Amidation Modifications