Protein Sequencing Service

The primary structure of a protein refers to the linear sequence of amino acids that make up its polypeptide chain. Protein sequencing, protein sequence analysis, protein primary structure analysis, and protein amino acid sequence analysis are all processes that determine the full amino acid sequence of a protein. The information obtained from protein sequencing has numerous valuable applications, such as: 1. Protein identification; 2. Designing probes for molecular cloning; 3. Synthesizing peptides for use as immunogens.

 

Protein sequencing services typically use two main methods: Edman degradation and mass spectrometry (MS). Currently, MS is the most widely used method for protein sequencing and identification, while Edman degradation remains crucial for characterizing protein N-termini. MtoZ Biolabs employs nano liquid chromatography-tandem mass spectrometry (nano LC-MS/MS ) and the Shimadzu's Edman degradation system for protein sequence analysis, offering services that include amino acid composition analysis, N-terminal sequencing, C-terminal sequencing, and full sequence analysis. For proteins with unknown sequences, de novo sequencing services are available.

 

Services at MtoZ Biolabs

1. MS-based Protein Sequence Analysis

MS offers advantages over Edman degradation, including higher sensitivity, faster peptide cleavage, and the ability to identify terminally blocked or modified proteins. MtoZ Biolabs utilizes advanced high-resolution MS platforms to provide comprehensive protein sequence analysis, ensuring full coverage of target protein sequences. This method is effective for confirming complete expression of recombinant proteins and detecting potential cleavages during expression.

 

Typically, trypsin is the sole enzyme used for protein sequence analysis, resulting in approximately 60% peptide coverage. To achieve complete sequence information, MtoZ Biolabs employs six proteases (trypsin, chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N) for enzymatic cleavage and MS. This approach, combined with advanced instrumentation like the Thermo Obitrap Fusion Lumos mass spectrometer, ensures high resolution and sensitivity, particularly for low-abundance peptides. The HCD and ETD fragmentation modes maintain peptide fragment integrity, enabling comprehensive N-terminal, C-terminal, and full-length sequence analysis.

 

2. Edman Degradation for N-terminal Protein Sequence Analysis

The Edman degradation method, developed by Pehr Edman in the early 1950s, is a chemical technique for obtaining N-terminal amino acid sequence information. This method labels and analyzes N-terminal amino acids without disrupting peptide bonds.

 

Edman degradation is widely used for N-terminal sequence analysis but has limitations, such as its inability to analyze proteins with blocked or chemically modified N-termini. In such cases, advanced nano LC-MS/MS platforms can be used for N-terminal sequence analysis, complementing Edman degradation. MtoZ Biolabs uses the Shimadzu's Edman sequencing system to provide N-terminal sequencing services for purified proteins, vaccines, and various protein samples, capable of determining up to 30 N-terminal amino acids.

 

Sample Submission Requirements

1. Sample Preparation

There are multiple methods for sample preparation, each with its own advantages and disadvantages. The optimal method can be determined based on laboratory experience and specific requirements for yield, purity, cost, and speed. Common Methods for Preparing Protein Sequencing Samples including:

(1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

(2) High-performance liquid chromatography (HPLC)

(3) Capillary electrophoresis (CE)

When selecting a sample preparation method, consider factors such as experiment duration, sample purity requirements, and the quantity of protein needed. If you lack the facilities for sample preparation, MtoZ Biolabs offers comprehensive protein analysis services, including sample preparation.

 

2. Sample Type and Transportation

MtoZ Biolabs' comprehensive protein sequencing services can analyze samples from PVDF membranes, gel spots, gel strips, solutions, etc. For transportation, tightly seal cut protein strips and ship with ice packs. Solution samples can be vacuum-dried or freeze-dried and transported with ice packs or dry ice. Please contact us for further information.