Quality Control Analysis of Proteomics MS/MS Data
Introduction

Data quality is a concern in proteomics experimental science. In this report, we assess the intrinsic features of the data set at different levels.

Methods and Data
Summary of Data Set

Table 1.  The Design of Experiment

sample bioRep techRep No. of fraction
55 1 1 2
55 1 2 2
75 1 1 2
75 1 2 2
Data Analysis

X!Tandem was used for analyzing the data. Parameters used in X!Tandem search were shown in Table 2. Protein identifications are inferred from peptide identifications, each protein identified having at least one associated unique peptide sequence identified at q-value equal or less than 0.01 (equivalent to a 1% FDR). The Occam's razor approach (Nesvizhskii, et al., 2003) was applied to deal with degenerate peptides by finding a minimum subset of proteins that covered all of the identified peptides.

Table 2.  The database search parameters

Item Value
Search engine X!Tandem
Enzyme trypsin
Fixed modifications Carbamidomethyl (C)
Variable modifications Oxidation (M)
Database target_decoy.fasta
Missed cleavages 0
Precursor mass error 10 ppm
Fragment mass error 0.6 Daltons
Results
Summary of protein identification

This part contains the basic statistics of MS/MS data.

Table 3.  Protein identification result for each fraction

sample bioRep techRep fraction spectrum_total spectrum peptide protein
55 1 1 5 1576 478(30.330%) 425 270
55 1 2 5 1562 456(29.193%) 415 256
55 1 1 6 1573 462(29.371%) 406 251
55 1 2 6 1558 487(31.258%) 448 294
75 1 1 5 1277 375(29.366%) 325 203
75 1 2 5 1256 350(27.866%) 308 192
75 1 1 6 1215 398(32.757%) 360 232
75 1 2 6 1208 361(29.884%) 327 218

Table 4.  Protein identification result for each technical replicate

sample bioRep techRep spectrum_total spectrum peptide protein
55 1 1 3149 940(29.851%) 807 453
55 1 2 3120 943(30.224%) 839 473
75 1 1 2492 773(31.019%) 659 371
75 1 2 2464 711(28.856%) 617 354

Table 5.  Protein identification result for each biological replicate

sample bioRep spectrum_total spectrum peptide protein
55 1 6269 1883(30.037%) 1458 700
75 1 4956 1484(29.944%) 1105 539
Summary charts

Summary plot for each technical replicate experiment.

Summary charts for sample: 55, biological: 1, technical: 1.

Figure 1.  Missed cleavages chart for sample: 55, biological replicate: 1, technical replicate: 1.

Figure 2.  Precursor ion charge chart for sample: 55, biological replicate: 1, technical replicate: 1.

Figure 3.  Peptide length chart for sample: 55, biological replicate: 1, technical replicate: 1.

Figure 4.  Precursor mass delta (Da) chart for sample: 55, biological replicate: 1, technical replicate: 1.

Figure 5.  Precursor mass delta (ppm) chart for sample: 55, biological replicate: 1, technical replicate: 1.

Figure 6.  Fragment ion mass delta (Da) chart for sample: 55, biological replicate: 1, technical replicate: 1.

Figure 7.  Unique spectrum per protein chart for sample: 55, biological replicate: 1, technical replicate: 1.

Figure 8.  Unique peptide per protein chart for sample: 55, biological replicate: 1, technical replicate: 1.

Figure 9.  Protein mass chart for sample: 55, biological replicate: 1, technical replicate: 1.

Summary charts for sample: 55, biological: 1, technical: 2.

Figure 10.  Missed cleavages chart for sample: 55, biological replicate: 1, technical replicate: 2.

Figure 11.  Precursor ion charge chart for sample: 55, biological replicate: 1, technical replicate: 2.

Figure 12.  Peptide length chart for sample: 55, biological replicate: 1, technical replicate: 2.

Figure 13.  Precursor mass delta (Da) chart for sample: 55, biological replicate: 1, technical replicate: 2.

Figure 14.  Precursor mass delta (ppm) chart for sample: 55, biological replicate: 1, technical replicate: 2.

Figure 15.  Fragment ion mass delta (Da) chart for sample: 55, biological replicate: 1, technical replicate: 2.

Figure 16.  Unique spectrum per protein chart for sample: 55, biological replicate: 1, technical replicate: 2.

Figure 17.  Unique peptide per protein chart for sample: 55, biological replicate: 1, technical replicate: 2.

Figure 18.  Protein mass chart for sample: 55, biological replicate: 1, technical replicate: 2.

Summary charts for sample: 75, biological: 1, technical: 1.

Figure 19.  Missed cleavages chart for sample: 75, biological replicate: 1, technical replicate: 1.

Figure 20.  Precursor ion charge chart for sample: 75, biological replicate: 1, technical replicate: 1.

Figure 21.  Peptide length chart for sample: 75, biological replicate: 1, technical replicate: 1.

Figure 22.  Precursor mass delta (Da) chart for sample: 75, biological replicate: 1, technical replicate: 1.

Figure 23.  Precursor mass delta (ppm) chart for sample: 75, biological replicate: 1, technical replicate: 1.

Figure 24.  Fragment ion mass delta (Da) chart for sample: 75, biological replicate: 1, technical replicate: 1.

Figure 25.  Unique spectrum per protein chart for sample: 75, biological replicate: 1, technical replicate: 1.

Figure 26.  Unique peptide per protein chart for sample: 75, biological replicate: 1, technical replicate: 1.

Figure 27.  Protein mass chart for sample: 75, biological replicate: 1, technical replicate: 1.

Summary charts for sample: 75, biological: 1, technical: 2.

Figure 28.  Missed cleavages chart for sample: 75, biological replicate: 1, technical replicate: 2.

Figure 29.  Precursor ion charge chart for sample: 75, biological replicate: 1, technical replicate: 2.

Figure 30.  Peptide length chart for sample: 75, biological replicate: 1, technical replicate: 2.

Figure 31.  Precursor mass delta (Da) chart for sample: 75, biological replicate: 1, technical replicate: 2.

Figure 32.  Precursor mass delta (ppm) chart for sample: 75, biological replicate: 1, technical replicate: 2.

Figure 33.  Fragment ion mass delta (Da) chart for sample: 75, biological replicate: 1, technical replicate: 2.

Figure 34.  Unique spectrum per protein chart for sample: 75, biological replicate: 1, technical replicate: 2.

Figure 35.  Unique peptide per protein chart for sample: 75, biological replicate: 1, technical replicate: 2.

Figure 36.  Protein mass chart for sample: 75, biological replicate: 1, technical replicate: 2.

Contaminants stat

The common Repository of Adventitious Proteins, cRAP (pronounced "cee-RAP"), is an attempt to create a list of proteins commonly found in proteomics experiments that are present either by accident or through unavoidable contamination of protein samples. The types of proteins included fall into three general classes:

  1. common laboratory proteins;

  2. proteins added by accident through dust or physical contact; and

  3. proteins used as molecular weight or mass spectrometry quantitation standards.

We added the cRAP database.

Table 6.  Identification of contaminant proteins

Accession Peptides Spectrum Sample ID Reason Class Description
K2C1_HUMAN 2 3 75_1_2 23 Human skin Dust/contact proteins (P04264) Keratin, type II cytoskeletal 1 (Cytokeratin-1) (CK-1) (Keratin-1) (K1) (67 kDa cytokeratin) (Hair alpha protein)
TRYP_PIG 1 1 75_1_1 13 Reagent Laboratory proteins (P00761) Trypsin precursor (EC 3.4.21.4)
UB2E1_HUMAN 1 1 75_1_1 111 UPS Sigma-Aldrich Universal Protein Standard (P51965) Ubiquitin-conjugating enzyme E2 E1 [UbcH6]
K2C1_HUMAN 2 2 55_1_2 23 Human skin Dust/contact proteins (P04264) Keratin, type II cytoskeletal 1 (Cytokeratin-1) (CK-1) (Keratin-1) (K1) (67 kDa cytokeratin) (Hair alpha protein)
TAU_HUMAN 1 1 55_1_2 106 UPS Sigma-Aldrich Universal Protein Standard (P10636) Microtubule-associated protein tau [Tau protein] 46,810 Recombinant E. coli 6-His
TRYP_PIG 2 2 55_1_1 13 Reagent Laboratory proteins (P00761) Trypsin precursor (EC 3.4.21.4)
AMYS_HUMAN 1 1 55_1_1 20 Human saliva Dust/contact proteins (P04745) Salivary alpha-amylase precursor (EC 3.2.1.1) (1,4-alpha-D-glucan glucanohydrolase)
Reproducibility
Reproducibility of result for each fraction

Reproducibility of total spectra for each fraction

Figure 37.  The distribution of the total spectra number

Figure 38.  Error bar plot of the total spectra number for each fraction

Reproducibility of identified spectra for each fraction

Figure 39.  The distribution of the identified spectra number

Figure 40.  Error bar plot of the identified spectra for each fraction

Reproducibility of identified peptides for each fraction

Figure 41.  The distribution of identified peptides number

Figure 42.  Error bar plot of the identified peptides for each fraction

Reproducibility of identified proteins for each fraction

Figure 43.  The distribution of the identified proteins number

Figure 44.  Error bar plot of the identified proteins for each fraction

Reproducibility of result for technical replicate

Figure 45.  Technical replicate of peptides' overlap between sample 55 biological replicate 1.

Figure 46.  Technical replicate of proteins' overlap between sample 55 biological replicate 1.

Figure 47.  Technical replicate of peptides' overlap between sample 75 biological replicate 1.

Figure 48.  Technical replicate of proteins' overlap between sample 75 biological replicate 1.

Reproducibility of result for different samples

Figure 49.  Peptides' overlap between different samples.

Figure 50.  Proteins' overlap between different samples.

Mass accuracy

Figure 51.  The mass error of the fragment ions

Figure 52.  The mass error of the precusor

Figure 53.  The mass error of the precusor

Separation efficiency
Fractionation accumulative effect

Figure 54.  Sample: 55 , biological replicate: 1 , technical replicate: 1, identified spectrum accumulative effect.

Figure 55.  Sample: 55 , biological replicate: 1 , technical replicate: 1, identified peptides accumulative effect.

Figure 56.  Sample: 55 , biological replicate: 1 , technical replicate: 1, identified proteins accumulative effect.

Figure 57.  Sample: 55 , biological replicate: 1 , technical replicate: 2, identified spectrum accumulative effect.

Figure 58.  Sample: 55 , biological replicate: 1 , technical replicate: 2, identified peptides accumulative effect.

Figure 59.  Sample: 55 , biological replicate: 1 , technical replicate: 2, identified proteins accumulative effect.

Figure 60.  Sample: 75 , biological replicate: 1 , technical replicate: 1, identified spectrum accumulative effect.

Figure 61.  Sample: 75 , biological replicate: 1 , technical replicate: 1, identified peptides accumulative effect.

Figure 62.  Sample: 75 , biological replicate: 1 , technical replicate: 1, identified proteins accumulative effect.

Figure 63.  Sample: 75 , biological replicate: 1 , technical replicate: 2, identified spectrum accumulative effect.

Figure 64.  Sample: 75 , biological replicate: 1 , technical replicate: 2, identified peptides accumulative effect.

Figure 65.  Sample: 75 , biological replicate: 1 , technical replicate: 2, identified proteins accumulative effect.