Navigation Links
Identification of Low-Level Oxidation Products in Calmodulin Using NanoSpray ,,, Ion Trap Mass Spectrometry

Andreas Hhmer, Helen Tran, and William S. Hancock

Protein Oxidation

The data presented here can be acquired using any Thermo Finnigan LCQTMion trap mass spectrometer with MSn capability.

Introduction

Calmodulin (CaM) is the primary high-affinity Ca2+-binding protein present in most eukaryotic cells. It regulates a variety of cellular Ca2+-dependent signaling pathways and plays a role in many cellular events, including protein phosphorylation. Ca2+binding induces a large conformational change in CaM that exposes methionine (Met) residues for interaction with CaM-binding enzymes that are otherwise inaccessible. CaM (MW 16706.40) has an unusual amino acid composition consisting of 148 amino acids with a high proportion of acidic residues (>34%) resulting in an isoelectric point around 3.9. CaM is particularly rich in Met residues, which account for more than 5% of its total composition, but it lacks significant amounts of other oxidatively sensitive amino acids such as cysteine, histidine and tryptophan. Evidence presented recently suggests that Met residues in CaM undergo an age-dependent modification to methionine sulfoxide (MetSO) in vivo1. The biological consequences of this oxidation can vary depending on the specific site of the oxidation: MetSO at the Nterminal end of CaM had an effect on CaM-dependent nitric oxide synthase (type-I) whereas MetSO near the C-terminus affected CaM-dependent cyclic phosphodiesterase. 2,3 For a range of biochemical studies, the detailed characterization of the age-related oxidative changes in CaM is important. Typically, this analysis involves Met-specific chemical cleavage (by cyanogen bromide, CNBr) or proteolytic digestion followed by mass spectrometry.

Goal

In this report, we describe the direct analysis of a CaM tryptic digest using a technique that shows:

1) The ability of MSn to quickly and easily identify MetSO-containing peptides.

2) The method for sequencing these peptides and determining the exact site of oxidation. (This is especially useful for peptides containing Met residues)

3) Complete characterization of oxidized CaM in a single experiment using less than 1 pmol of material.


Sequence and tryptic mapping of vertebrate CaM. The arrows indicate theoretical cleavage sites for trypsin, while the letters indicate tryptic fragments observed in oxidized CaM.

Experimental Conditions

Sample Preparation: A 40-M solution of High Purity Bovine Brain Calmodulin (Calbiochem) was oxidized in air-saturated sodium phosphate buffer (20 mM, pH 7.4), containing 100-mM KCl at room temperature and purified by HPLC on a HyPURITY C8 guard column (2 2 mm) (Thermo Hypersil-Keystone) equilibrated with 0.1% (v/v) aqueous formic acid and eluted with 80% AcN/20% H2O/0.1% formic acid (v/v). CaM samples were collected, dried under vacuum, re-dissolved in 50-mM Tris-HCl buffer (pH 8.2), and digested with sequencing-grade bovine pancreas trypsin for 12 hours at 37 C with a CaM:trypsin ratio of 100:1.

Sample Analysis: The CaM digest was separated on a 150 0.18 mm HyPURITY C8 column (The rmo Hypersil-Keystone) at a flow rate of 700 nL/min with a linear gradient of 0.7%/min from 3% Solvent B (100% AcN with 0.1% aqueous formic acid) to 45% B followed by a linear gradient of 1%/min to 80% B. Solvent A was 0.1% aqueous formic acid. The column flow rate was produced by splitting the primary flow rate of 80 L/min from a Surveyor HPLC system (Thermo Finnigan) at a ratio of 1:115.

A NanoSpray Ion Source (Thermo Finnigan) with a 30-m ID fused-silica PicoTip was used to introduce the column effluent into an LCQ Deca ion trap mass spectrometer using the following source conditions:

Capillary Temp: 175 C
Spray Voltage: 1.4kV
Capillary Voltage: 25 V
Tube Lens Offset: 0 V CaM mediates vital processes including inflammation, short-term
memory, the immune response and the cell cycle implicating it in
AIDS, Alzheimers, certain cancers, and other diseases.
Discussion

MS analysis of 900 fmoles of tryptic digest of oxidized CaM revealed 13 peptide fragments that were identified in a single Data Dependent fullscan MS/MS experiment using Dynamic Exclusion (Figure 1). The result from the MS/MS analysis shows coverage for a majority of the amino acid sequence of CaM. In addition to the expected native tryptic fragments, three peaks were identified as peptides containing MetSO (Table 1). Since sulfoxidation of Met residues in CaM plays a crucial role in the activation of target enzymes, the exact position and nature of the oxygen addition (+16 m/z) to the Met residues in the trypti c fragments T12 and T13 were further investigated by MS2 and MS3 experiments.

The peaks at retention times of 45.87 min and 49.34 min have m/z values corresponding to an oxidized form of T12. This peptide is of particular interest because it contains two possible oxidation sites, Met109 and Met124.


Figure 1. Base peak mass spectrum of tryptic fragments of oxidized CaM separated on a C8 Hypurity column and detected with Data Dependent ion trap MS/MS.


Table 1: Theoretical monoisotopic masses of peptide fragments compared to measured m/z values recovered from a tryptic digest of oxidized CaM by full-scan Data Dependent LC-MS/MS.

The full-scan MS2 spectrum of the doubly charged ion at m/z 1209.5 for the peak at RT = 45.87 min shows a series of corresponding y and b ions for a MetSO-containing T12 peptide (Figure 2A). The appearance of MetSO-specific neutral loss sequence ions (SOCH4) for the y ion series is consistent with the sulfoxide located on the Cterminal portion of the T12 peptide (see insert, Figure 2A). Analysis of the most abundant peak (m/z 1177.4) in the MS2 spectrum by MS3 confirms oxidation of Met-124 by detection of MetSOspecific neutral loss sequence ions of C-terminal origin (Figure 2b).


Figure 2A. Full-scan MS2 spectrum of doubly-charged ion at m/z 1209.5 for peak at RT 45.87 min.
Insert: Amino acid sequence of T12 fragment indicating major C- and Nterminal fragment ions detected by full-scan MS2.


Figure 2B. Full-scan MS3 spectrum of doubly-charged ion at m/z 1177.5 for peak at RT 45.87 min.

In contrast, the MS2 spectrum of the doubly charged ion at m/z 1209.5 for the peak at RT = 49.45 min includes y and b-series ions, as well as the MetSO associated neutral loss for the b ions, indicating that the MetSO is located at the Nterminus of the peptide, e.g., Met109 (Figure 3A and Figure 3A insert). MS3 analysis of the peak at m/z 1177.4 in the MS2 spectrum substantiates oxidation of Met109 through the detection of MetSO-specific neutral loss sequence ions of Nterminal origin (Figure 3B).

Similarly, MS2 and MS3 experiments were conducted for the peak at RT = 56.31, and it was established that the corresponding peptide contained MetSO exclusively at Met144. The extent and character of MetSO formation in CaM shown in this experiment is in agreement with results published previously2.


Figure 3A. Full-scan MS2 spectrum of doubly-charged ion at m/z 1209.5 for peak at RT 49.45 min.
Insert: Amino acid sequence of T12 fragment indicating major C- and Nterminal fragment ions detected by full-scan MS2.




Figure 3B. Full-scan MS3 spectrum of doubly-charged ion at m/z 1177.5 for peak at RT 49.45 min.

Conclusions

The identification of low-level oxidation products such as MetSO in proteins and peptides can be easily achieved using any Thermo Finnigan LCQ series ion trap mass spectrometer with MSn capability. The combination of D ata Dependent Dynamic Exclusion and MSn has allowed definitive characterization of peptide sequence and oxidation products in a single, rapid experiment, illustrating the power of the ion trap for the identification of post-translational modifications.

References

Gao, J.; Yin, D.H.; Yao, Y.; Williams, T.D.; Squier, T.C. Progressive decline in the ability of calmodulin isolated from aged brain to activate the plasma membrane Ca2-ATPase. Biochemistry 37: 9536-9548; 1998.
Hhmer, A.F.R.; (1997) Peroxynitrite Mediated Methionine Oxidation of Calmodulin: Chemistry and Biological Consequences, Ph.D. Thesis, The University of Kansas.
Walsh, M.; Stevens, F.C. Chemical Modification Studies on the Ca2-Dependent Protein Modulator: The Role of Methionine Residues in the Activation of Cyclic Nucleotide Phosphodiesterase. Biochemistry 17: 3924-3928; 1978.






'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Identification of Differentially Expressed Gene Products with the CastAway System*
2. An Optimal DNA Microarray Substrate for the Identification of Fetal and Somatic Liver Stem Cells of the Rat
3. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
4. Protect RNA and Improve Target Cell Identification
5. Identification of Disease Causing Mutations in Phenylketonuria by Denaturing Gradient Gel Electrophoresis Using the DCode System
6. Identification of Nonspecific Products Using Melt-Curve Analysis on the iCycler iQ Detection System, Rev A
7. Identification of cells in S phase using the Cell Proliferation Fluorescence Kit and IN Cell Analyzer 1000
8. Identification of Insecticides and Herbicides by LC/MS/MS Using the API 2000 LC/MS/MS System
9. Identification of Phase I and Phase II Metabolites of Buspirone on the Q TRAP LC/MS/MS System
10. ESR1 Gene Expression in Oncology and Metabolic Diseases Using the ASCENTATM System Target Validation and Identification of Novel Disease Indications
11. High-Throughput System Generates Ultra-Pure PCR Products Suitable for All Applications
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... ... June 27, 2016 , ... Parallel 6 , the ... today the Clinical Reach Virtual Patient Encounter CONSULT module which enables both ... physician and clinical trial team. , Using the CONSULT module, patients and physicians can ...
(Date:6/27/2016)... -- Liquid Biotech USA , Inc. ... Research Agreement with The University of Pennsylvania ("PENN") ... patients.  The funding will be used to assess ... outcomes in cancer patients undergoing a variety of ... to support the design of a therapeutic, decision-making ...
(Date:6/24/2016)... 24, 2016 Epic Sciences unveiled a ... susceptible to PARP inhibitors by targeting homologous recombination ... The new test has already been incorporated into ... cancer types. Over 230 clinical trials ... pathways, including PARP, ATM, ATR, DNA-PK and WEE-1. ...
(Date:6/23/2016)... ... June 23, 2016 , ... Mosio, a leader in ... Trials Patient Recruitment and Retention Tips.” Partnering with experienced clinical research professionals, Mosio ... practical tips, tools, and strategies for clinical researchers. , “The landscape of how ...
Breaking Biology Technology:
(Date:6/2/2016)... 2, 2016 The Department of Transport ... the 44 million US Dollar project, for the , ... including Personalization, Enrolment, and IT Infrastructure , to ... production and implementation of Identity Management Solutions. Numerous renowned international ... Decatur was selected for the most compliant ...
(Date:6/1/2016)... YORK , June 1, 2016 ... Technology in Election Administration and Criminal Identification to Boost ... to a recently released TechSci Research report, " Global ... By Region, Competition Forecast and Opportunities, 2011 - 2021", ... 24.8 billion by 2021, on account of growing security ...
(Date:5/16/2016)... May 16, 2016   EyeLock LLC , a ... the opening of an IoT Center of Excellence in ... expand the development of embedded iris biometric applications. ... of convenience and security with unmatched biometric accuracy, making ... aside from DNA. EyeLock,s platform uses video technology to ...
Breaking Biology News(10 mins):