diff --git a/src/main/java/proteomics/Index/BuildIndex.java b/src/main/java/proteomics/Index/BuildIndex.java
index d0d8c25..0d0c143 100644
--- a/src/main/java/proteomics/Index/BuildIndex.java
+++ b/src/main/java/proteomics/Index/BuildIndex.java
@@ -99,7 +99,7 @@ public BuildIndex(Map<String, String> parameter_map) throws Exception {
         }
 
         // define a new MassTool object
-        mass_tool_obj = new MassTool(missed_cleavage, fix_mod_map, parameter_map.get("cleavage_site").trim(), parameter_map.get("protection_site").trim(), parameter_map.get("is_from_C_term").trim().contentEquals("1"), mz_bin_size * 0.5, one_minus_bin_offset, "N14");
+        mass_tool_obj = new MassTool(missed_cleavage, fix_mod_map, parameter_map.get("cleavage_site_1").trim(), parameter_map.get("protection_site_1").trim(), parameter_map.get("is_from_C_term_1").trim().contentEquals("1"), parameter_map.getOrDefault("cleavage_site_2", null), parameter_map.getOrDefault("protection_site_2", null), parameter_map.containsKey("is_from_C_term_2") ? parameter_map.get("is_from_C_term_2").trim().contentEquals("1") : null, mz_bin_size * 0.5, one_minus_bin_offset, "N14");
 
         // generate seq_pro_map
         Map<String, boolean[]> seq_term_map = new HashMap<>();
@@ -139,7 +139,7 @@ public BuildIndex(Map<String, String> parameter_map) throws Exception {
             boolean proteinCTerm = seq_term_map.get(seq)[1];
 
             // mod free
-            Set<Short> linkSiteSet = getLinkSiteSet(seq, parameter_map.get("is_from_C_term").trim().contentEquals("1"), parameter_map.get("cleavage_site"), proteinNTerm, proteinCTerm, linker_type);
+            Set<Short> linkSiteSet = getLinkSiteSet(seq, parameter_map.get("is_from_C_term_1").trim().contentEquals("1"), parameter_map.get("cleavage_site_1"), parameter_map.containsKey("is_from_C_term_2") ? parameter_map.get("is_from_C_term_2").trim().contentEquals("1") : null, parameter_map.getOrDefault("cleavage_site_2", null), proteinNTerm, proteinCTerm, linker_type);
             if (!linkSiteSet.isEmpty()) {
                 double totalMass = (mass_tool_obj.calResidueMass(seq) + mass_tool_obj.H2O);
                 int bin = massToBin(totalMass);
@@ -548,28 +548,52 @@ private Set<String> checkKCTermMod(Set<String> varSeqSet) { // eliminate those s
         }
     }
 
-    private Set<Short> getLinkSiteSet(String seq, boolean is_from_C_term, String cleavage_site, boolean n_term, boolean c_term, short linker_type) {
+    private Set<Short> getLinkSiteSet(String seq, boolean is_from_C_term_1, String cleavage_site_1, Boolean is_from_C_term_2, String cleavage_site_2, boolean n_term, boolean c_term, short linker_type) {
         AA[] aa_list = MassTool.seqToAAList(seq);
         Set<Short> output = new HashSet<>(5, 1);
         for (int i = 1; i < aa_list.length - 1; ++i) {
             if (linker_type == 1 && aa_list[i].aa == 'K' && (Math.abs(aa_list[i].ptmDeltaMass) < varModMassResolution)) {
-                if (is_from_C_term) {
-                    if (i < aa_list.length - 2 || !cleavage_site.contains("K")) {
+                if (is_from_C_term_2 == null) {
+                    if (is_from_C_term_1 && (i < aa_list.length - 2 || !cleavage_site_1.contains("K"))) {
+                        output.add((short) i);
+                    } else if (!is_from_C_term_1 && (i > 1 || !cleavage_site_1.contains("K"))) {
                         output.add((short) i);
                     }
                 } else {
-                    if (i > 1 || !cleavage_site.contains("K")) {
-                        output.add((short) i);
+                    if (is_from_C_term_1 && (i < aa_list.length - 2 || !cleavage_site_1.contains("K"))) {
+                        if (is_from_C_term_2 && (i < aa_list.length - 2 || !cleavage_site_2.contains("K"))) {
+                            output.add((short) i);
+                        } else if (!is_from_C_term_2 && (i > 1 || !cleavage_site_2.contains("K"))) {
+                            output.add((short) i);
+                        }
+                    } else if (!is_from_C_term_1 && (i > 1 || !cleavage_site_1.contains("K"))) {
+                        if (is_from_C_term_2 && (i < aa_list.length - 2 || !cleavage_site_2.contains("K"))) {
+                            output.add((short) i);
+                        } else if (!is_from_C_term_2 && (i > 1 || !cleavage_site_2.contains("K"))) {
+                            output.add((short) i);
+                        }
                     }
                 }
             } else if (linker_type == 2 && aa_list[i].aa == 'C' && (Math.abs(aa_list[i].ptmDeltaMass) < varModMassResolution)) {
-                if (is_from_C_term) {
-                    if (i < aa_list.length - 2 || !cleavage_site.contains("C")) {
+                if (is_from_C_term_2 == null) {
+                    if (is_from_C_term_1 && (i < aa_list.length - 2 || !cleavage_site_1.contains("C"))) {
+                        output.add((short) i);
+                    } else if (!is_from_C_term_1 && (i > 1 || !cleavage_site_1.contains("C"))) {
                         output.add((short) i);
                     }
                 } else {
-                    if (i > 1 || !cleavage_site.contains("C")) {
-                        output.add((short) i);
+                    if (is_from_C_term_1 && (i < aa_list.length - 2 || !cleavage_site_1.contains("C"))) {
+                        if (is_from_C_term_2 && (i < aa_list.length - 2 || !cleavage_site_2.contains("C"))) {
+                            output.add((short) i);
+                        } else if (!is_from_C_term_2 && (i > 1 || !cleavage_site_2.contains("C"))) {
+                            output.add((short) i);
+                        }
+                    } else if (!is_from_C_term_1 && (i > 1 || !cleavage_site_1.contains("C"))) {
+                        if (is_from_C_term_2 && (i < aa_list.length - 2 || !cleavage_site_2.contains("C"))) {
+                            output.add((short) i);
+                        } else if (!is_from_C_term_2 && (i > 1 || !cleavage_site_2.contains("C"))) {
+                            output.add((short) i);
+                        }
                     }
                 }
             }
diff --git a/src/main/java/proteomics/Parameter/Parameter.java b/src/main/java/proteomics/Parameter/Parameter.java
index d476cab..bd8129f 100644
--- a/src/main/java/proteomics/Parameter/Parameter.java
+++ b/src/main/java/proteomics/Parameter/Parameter.java
@@ -41,10 +41,17 @@ public Parameter(String parameterFile) throws Exception {
                     } else {
                         Matcher enzymeMatcher = enzymePattern.matcher(line);
                         if (enzymeMatcher.matches()) {
-                            parameterMap.put("enzyme_name", enzymeMatcher.group(1).trim());
-                            parameterMap.put("is_from_C_term", enzymeMatcher.group(2).trim());
-                            parameterMap.put("cleavage_site", enzymeMatcher.group(3).trim());
-                            parameterMap.put("protection_site", enzymeMatcher.group(4).trim());
+                            if (parameterMap.containsKey("enzyme_name_1")) {
+                                parameterMap.put("enzyme_name_2", enzymeMatcher.group(1).trim());
+                                parameterMap.put("is_from_C_term_2", enzymeMatcher.group(2).trim());
+                                parameterMap.put("cleavage_site_2", enzymeMatcher.group(3).trim());
+                                parameterMap.put("protection_site_2", enzymeMatcher.group(4).trim());
+                            } else {
+                                parameterMap.put("enzyme_name_1", enzymeMatcher.group(1).trim());
+                                parameterMap.put("is_from_C_term_1", enzymeMatcher.group(2).trim());
+                                parameterMap.put("cleavage_site_1", enzymeMatcher.group(3).trim());
+                                parameterMap.put("protection_site_1", enzymeMatcher.group(4).trim());
+                            }
                         }
                     }
                 }
diff --git a/src/main/resources/parameter.def b/src/main/resources/parameter.def
index 0368347..ae3aeb1 100644
--- a/src/main/resources/parameter.def
+++ b/src/main/resources/parameter.def
@@ -66,7 +66,7 @@ O = 0
 n = 0
 c = 0
 
-# Enzyme digestion specifications. Only support one enzyme at a time.
+# Enzyme digestion specifications. Support up to two enzymes simultaneously.
 # enzyme name  	 is cut from C-term 	 cleavage site 	 protection site
 Trypsin        	 1                  	 KR            	 P
 # Trypsin/P      1                  	 KR            	 -