Molecular Weight Calculator Expasy

Calculate and understand the molecular weight of proteins and peptides.

Protein Molecular Weight Calculator

Enter the amino acid sequence to estimate the molecular weight. This tool utilizes average isotopic masses.

Amino Acid Residue Masses

Amino Acid (1-Letter)	Full Name	Residue Mass (Da)
A	Alanine	71.0788
R	Arginine	156.1875
N	Asparagine	114.1039
D	Aspartic Acid	115.0886
C	Cysteine	103.1389
E	Glutamic Acid	129.1155
Q	Glutamine	128.1314
G	Glycine	57.0519
H	Histidine	137.1411
I	Isoleucine	113.1594
L	Leucine	113.1594
K	Lysine	128.1741
M	Methionine	131.1926
F	Phenylalanine	147.1766
P	Proline	97.1167
S	Serine	87.0782
T	Threonine	101.1051
W	Tryptophan	174.2017
Y	Tyrosine	163.1760
V	Valine	99.1326

Molecular Weight Distribution

What is the EXpasy Molecular Weight Calculator?

The EXpasy Molecular Weight Calculator is a specialized online tool designed to accurately determine the theoretical molecular weight (MW) of peptides and proteins based on their amino acid sequence. Proteins are fundamental macromolecules in all living organisms, playing crucial roles in virtually every biological process. Understanding their molecular weight is essential for various biochemical, molecular biology, and proteomics applications. This tool, often found on platforms like the SIB Swiss Institute of Bioinformatics' EXpasy portal, simplifies this calculation, providing a vital piece of information for researchers.

Who Should Use It:

• Biochemists and Molecular Biologists: For characterizing newly synthesized proteins, verifying protein identity, and understanding protein function.
• Proteomics Researchers: Essential for mass spectrometry analysis, where precise mass measurements are used to identify and quantify proteins.
• Drug Discovery Scientists: When designing peptide-based therapeutics or analyzing protein interactions.
• Students and Educators: As a learning tool to grasp the relationship between amino acid composition and protein mass.

Common Misconceptions:

• Exact vs. Average Mass: This calculator typically uses average isotopic masses for elements (e.g., Carbon ~12.011 Da, Hydrogen ~1.008 Da). In reality, proteins exist as a mixture of isotopologues (molecules with different isotopic compositions), especially when considering the exact mass of specific isotopes.
• Post-Translational Modifications (PTMs): The standard calculator does not account for PTMs like phosphorylation, glycosylation, or methylation, which significantly alter the protein's mass. Specialized calculators or manual adjustments are needed for PTMs.
• N-terminal Modification: While the calculation subtracts water for peptide bonds, it might not always explicitly account for N-terminal modifications like methionine cleavage if not specified.

Molecular Weight Calculator (EXpasy) Formula and Mathematical Explanation

The calculation of a protein's molecular weight from its amino acid sequence is based on the fundamental principles of peptide bond formation and the atomic composition of amino acids. When two amino acids join to form a peptide bond, a molecule of water (H₂O) is released. Therefore, the mass of a polypeptide chain is the sum of the masses of its constituent amino acid residues minus the mass of the water molecules lost during bond formation.

Step-by-Step Derivation:

1. Identify Amino Acid Residues: The primary input is the amino acid sequence, represented by single-letter codes.
2. Determine Residue Masses: Each amino acid has a specific average mass when it exists as a residue within a polypeptide chain. This is calculated by taking the mass of the free amino acid and subtracting the mass of one water molecule (H₂O).
3. Sum Residue Masses: Add up the individual masses of all the amino acid residues in the sequence.
4. Account for Water Loss: For a polypeptide chain composed of 'n' amino acid residues, 'n-1' peptide bonds are formed. Each peptide bond formation releases one water molecule.
5. Calculate Total Molecular Weight: The final molecular weight is the sum of all residue masses minus the total mass of the released water molecules.

Formula:

MW = (Σ Mass(Residue_i)) - (n - 1) * Mass(H₂O)

Where:

• MW = Molecular Weight of the polypeptide
• Σ Mass(Residue_i) = Sum of the average masses of each amino acid residue in the sequence (from i=1 to n)
• n = Number of amino acid residues in the sequence
• Mass(H₂O) = Average mass of a water molecule (approximately 18.015 Da)

Variables Table:

Variable	Meaning	Unit	Typical Range/Note
Amino Acid Sequence	The chain of amino acids making up the protein/peptide.	N/A (String)	Standard 20 amino acids (e.g., MGYTWY)
n (Number of Residues)	The total count of amino acids in the sequence.	Count	≥ 1
Mass(Residue_i)	The average mass of a specific amino acid residue.	Daltons (Da)	Approx. 57.05 (Glycine) to 174.20 (Tryptophan)
Mass(H₂O)	The average mass of a water molecule.	Daltons (Da)	~18.015 Da
MW	The calculated molecular weight of the peptide/protein.	Daltons (Da)	Variable, depends on sequence length and composition

Practical Examples (Real-World Use Cases)

Example 1: Calculating the MW of a Small Peptide Hormone

Scenario: A researcher is studying a small peptide hormone, Oxytocin, with the sequence: CYIQNCPLG.

Inputs:

• Amino Acid Sequence: CYIQNCPLG

Calculation Steps (Conceptual):

1. Count Residues: There are 9 amino acid residues (n=9).
2. Sum Residue Masses:
   • C (Cysteine): 103.1389 Da
   • Y (Tyrosine): 163.1760 Da
   • I (Isoleucine): 113.1594 Da
   • Q (Glutamine): 128.1314 Da
   • N (Asparagine): 114.1039 Da
   • C (Cysteine): 103.1389 Da
   • P (Proline): 97.1167 Da
   • L (Leucine): 113.1594 Da
   • G (Glycine): 57.0519 Da
   • Total Sum: 1092.0779 Da
3. Account for Water Loss: Number of bonds = n – 1 = 9 – 1 = 8. Mass lost = 8 * 18.015 Da = 144.120 Da.
4. Calculate MW: 1092.0779 Da – 144.120 Da = 947.9579 Da.

Calculator Output (Approximate):

• Number of Residues: 9
• Mass of Residues: 1092.08 Da
• Mass of Water (H₂O): 144.12 Da
• Molecular Weight: 947.96 Da

Interpretation: The calculated molecular weight of Oxytocin is approximately 947.96 Daltons. This value is crucial for experiments involving its separation, purification, or detection, such as gel electrophoresis or mass spectrometry.

Example 2: Calculating the MW of a larger protein fragment

Scenario: A researcher analyzes a protein digest and identifies a fragment with the sequence: MKTIIALVG.

Inputs:

• Amino Acid Sequence: MKTIIALVG

Calculation Steps (Conceptual):

1. Count Residues: There are 8 amino acid residues (n=8).
2. Sum Residue Masses:
   • M (Methionine): 131.1926 Da
   • K (Lysine): 128.1741 Da
   • T (Threonine): 101.1051 Da
   • I (Isoleucine): 113.1594 Da
   • I (Isoleucine): 113.1594 Da
   • A (Alanine): 71.0788 Da
   • L (Leucine): 113.1594 Da
   • V (Valine): 99.1326 Da
   • Total Sum: 870.9994 Da
3. Account for Water Loss: Number of bonds = n – 1 = 8 – 1 = 7. Mass lost = 7 * 18.015 Da = 126.105 Da.
4. Calculate MW: 870.9994 Da – 126.105 Da = 744.8944 Da.

Calculator Output (Approximate):

• Number of Residues: 8
• Mass of Residues: 871.00 Da
• Mass of Water (H₂O): 126.11 Da
• Molecular Weight: 744.89 Da

Interpretation: The molecular weight of this protein fragment is approximately 744.89 Da. This information aids in identifying this specific fragment during mass spectrometry analysis, comparing experimental results against theoretical predictions.

How to Use This Molecular Weight Calculator (EXpasy)

Using this molecular weight calculator is straightforward and requires minimal input. Follow these steps to obtain the molecular weight of your peptide or protein sequence:

1. Enter Amino Acid Sequence: In the "Amino Acid Sequence" input field, type the sequence of your peptide or protein using the standard single-letter codes for the 20 common amino acids (e.g., A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V). Ensure accuracy and correct order.
2. Click "Calculate": Once the sequence is entered, click the "Calculate" button. The calculator will process the sequence instantly.
3. Review Results: The results section will display:
   • Primary Highlighted Result: The calculated Molecular Weight (MW) in Daltons (Da), prominently displayed.
   • Intermediate Values: The total number of residues, the sum of individual residue masses before water subtraction, and the total mass of water subtracted.
   • Formula Explanation: A brief description of the calculation method used.
4. Use "Copy Results": If you need to save or share the results, click the "Copy Results" button. This will copy the main MW, intermediate values, and key assumptions (like using average isotopic masses) to your clipboard.
5. Use "Reset": To clear the current input and results and start over, click the "Reset" button. It will restore the default state.

Decision-Making Guidance:

• Experimental Verification: Compare the calculated theoretical MW with experimental MW values obtained from techniques like mass spectrometry. Significant deviations may indicate post-translational modifications, unusual amino acids, or calculation errors.
• Troubleshooting: If your experimental MW doesn't match the theoretical one, re-check the amino acid sequence for errors and consider potential modifications not accounted for by this basic calculator.
• Design: This tool is invaluable during the design phase of synthetic peptides to predict their properties.

Key Factors That Affect Molecular Weight Results

While the EXpasy molecular weight calculator provides a theoretical value based on the amino acid sequence, several biological and chemical factors can lead to discrepancies between theoretical and experimentally determined molecular weights. Understanding these factors is crucial for accurate interpretation in a biological context.

• Amino Acid Sequence Accuracy: The most fundamental factor. Any errors in transcribing or inputting the amino acid sequence directly lead to an incorrect theoretical molecular weight. This is the primary variable controlled by the user.
• Isotopic Composition: The calculator uses average isotopic masses (e.g., Carbon-12, Carbon-13). However, proteins exist as a mixture of molecules with different isotopic compositions (isotopologues). Mass spectrometry often measures the monoisotopic mass (using the most abundant isotope for each atom) or the average mass, and the specific measurement method impacts the observed value.
• Post-Translational Modifications (PTMs): This is a major factor for many proteins. PTMs such as phosphorylation (+79.966 Da), glycosylation (variable and significant mass addition), acetylation (+42.011 Da), methylation (+14.016 Da), ubiquitination (very large mass addition), and disulfide bond formation (-2.016 Da per bond, due to loss of 2H) drastically alter the final mass of a protein. This calculator does not account for PTMs.
• N-terminal and C-terminal Modifications: Beyond peptide bond formation, the N-terminus might be acetylated or formylated, and the C-terminus might be amidated. These modifications add specific masses not included in the basic calculation. Signal peptide cleavage, if not accounted for in the provided sequence, also affects the final processed protein's mass.
• Amino Acid Cyclization: Certain amino acids, particularly at the N-terminus, can cyclize (e.g., N-terminal pyroglutamate formation from Glutamine), leading to a loss of ~18 Da (water molecule).
• Chemical Degradation or Fragmentation: In experimental settings, proteins or peptides can undergo partial degradation or fragmentation, leading to observed masses that are lower than the intact theoretical mass.
• Salt Bridges and Non-Covalent Interactions: While these don't directly change the *mass* of the molecule, they influence its conformation and interactions, which can indirectly affect how its mass is measured or interpreted in certain complex assays.
• Buffer Composition and pH: The ionization state of amino acid side chains (especially acidic and basic ones) is affected by pH. While this doesn't change the fundamental mass, it impacts the charge state, which is critical for techniques like mass spectrometry.

Frequently Asked Questions (FAQ)

What is a Dalton (Da)?

A Dalton (Da) is a unit of mass commonly used in biochemistry and molecular biology, approximately equal to the mass of one hydrogen atom. It's often used interchangeably with the atomic mass unit (amu). For proteins and peptides, it's a convenient unit for expressing molecular weight.

Does the calculator account for PTMs?

No, this calculator provides the theoretical molecular weight based solely on the standard amino acid sequence and average isotopic masses. It does not account for post-translational modifications (PTMs) like phosphorylation, glycosylation, etc., which significantly alter the actual mass of a protein.

What is the difference between average mass and monoisotopic mass?

Average mass is the weighted average of the masses of all naturally occurring isotopes of the elements in a molecule. Monoisotopic mass is the mass of a molecule calculated using the mass of the most abundant isotope for each atom (e.g., 12C, 1H, 14N, 16O, 32S). Mass spectrometry can measure both, and they differ slightly.

Why is the mass of water subtracted?

When amino acids link together to form a peptide bond, a molecule of water (H₂O) is released. For a polypeptide chain of 'n' amino acids, 'n-1' water molecules are lost. Subtracting the mass of these water molecules gives the correct mass of the resulting peptide chain.

Can I input non-standard amino acids?

This calculator is designed for the 20 standard proteinogenic amino acids using their single-letter codes. Inputting non-standard amino acids or other characters may lead to incorrect results or errors.

What happens if I enter a single amino acid?

If you enter a single amino acid, the formula correctly calculates `(Mass(Residue)) – (1 – 1) * Mass(H₂O)`, which simplifies to just the residue mass. This is the expected molecular weight for a free amino acid residue.

How accurate are the results?

The results are theoretically accurate based on the standard atomic masses and the formula for peptide bond formation. However, experimental conditions and biological reality (PTMs, isotopic variations) mean the actual measured mass might differ.

Is this calculator the same as the Expasy ProtParam tool?

The EXpasy ProtParam tool calculates a wider range of protein properties, including molecular weight, isoelectric point, amino acid composition, and more. This calculator specifically focuses on the molecular weight calculation as a core function, inspired by the foundational aspects of tools like ProtParam.